Endophytic yeast strains, methods for ethanol and xylitol production, methods for biological nitrogen fixation, and a genetic source for improvement of industrial strains

ABSTRACT

The present invention provides novel endophytic yeast strains capable of metabolizing both pentose and hexose sugars. Methods of producing ethanol and xylitol using the novel endophytic yeast are provided herein. Also provided are methods of fixing nitrogen and fertilizing a crop using the novel endophytic yeast strains provided herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a U.S. National Phase Application under 35U.S.C. §371 of International Application No. PCT/US2010/027234, filed onMar. 12, 2010, which claims the benefit of U.S. Provisional ApplicationNo. 61/160,077, filed Mar. 13, 2009, expressly incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

This application includes a Sequence Listing as an ASCII text file named“-86-1.txt” created Nov. 2, 2011, machine format IBM-PC, MS-Windowsoperating system, and containing 229,376 bytes. The material containedin this text file is incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Endophytic microorganisms occur within living plant tissues withoutcausing apparent damage to the host (Petrini, 1991). To date, endophyticyeasts have been isolated from a variety of plants, including roots ofZea mays L. (maize) (Nassar et al., 2005) and roots of Musa acuminate L.(banana) (Cao et al., 2002), and leaves of Oryza sativa L. (rice) (Tianet al., 2004), Solanum lycopersicum L. (tomato) (Larran et al., 2001),and Triticum aestivum L. (wheat) (Larran et al., 2002). In a series ofstudies on endophytic microorganisms in wild and hybrid Populus species,(Doty et al., 2005; Doty et al., 2009) three yeast strains wereisolated.

Identification of yeast species from morphological and physiologicalcharacteristics has been complemented with and improved by molecularmethods in the last 20 years. Analyses of small subunit (18 S) ribosomalRNA (rRNA) gene sequences, extremely important in phylogenetic analysesof species in bacteria, generally are not adequate to differentiateyeast species (James et al., 1996; Kurtzman and Robnett, 2003).Sequencing domains 1 and 2 (D1/D2) of large subunit (26S) rRNA gene havebeen used by many researchers to determine yeast species because thisapproach is rapid and effective, and a large number of sequences areavailable for comparison in online databases (Kurtzman and Robnett,1998; Fell et al., 2000; Kurtzman, 2006). The internal transcribedspacer (ITS) regions ITS1 and ITS2, flanking the 5.8S gene of rRNA, arealso highly substituted and are used for yeast identification. Scorzettiet al. (2002) found that analyzing ITS sequences allowed them to detectspecies among basidiomycetous species more effectively than using D1/D2.For example, Sporobolomyces holsaticus Windisch ex Yarrow & Fell andSporidiobolus johnsonii Nyland are identical in D1/D2 sequences,register 93% DNA hybridization (Boekhout, 1991), and differ in five basepositions in the ITS sequences. In contrast, Rhodotorula glutinis(Fresen.) F. C. Harrison and Rhodotorula graminis Di Menna are identicalin the ITS region but differ in one base position in D1/D2; they areconsidered to be separate species based on 35-40% DNA hybridization(Gadanho and Sampaio, 2002). Consequently, it appears useful to sequenceboth D1/D2 and ITS regions when distinguishing closely related species,while defining species taxonomically requires classical phenotypicinformation (Scorzetti et al., 2002).

Recently, the role of endophytic microorganisms in the promotion ofplant growth has received increased attention. Endophytes can promoteplant growth through different mechanisms, including delivery of fixednitrogen to host plants, production of plant growth regulators, andbiological control of plant pathogens (Ryan et al., 2008). Endophyticyeast strains have been shown to be able to promote the growth of maize(Nassar et al., 2005) and Beta vulgaris L. (sugar beet) (El-Tarabily,2004) by producing plant auxins, such as indole-3-acetic acid (IAA) andindole-3-pyruvic acid (IPYA) (Nassar et al., 2005).

Efficient industrial production of biofuels, such as bioethanol, holdspromise for serving the growing energy needs of the world in the nearfuture. Ethanolic fermentation of cellulosic and lignocellulosic biomassby microorganisms such as yeast is currently employed in the industrialproduction of bioethanol. However, the lack of non-pathogenicmicroorganisms that efficiently metabolize both five carbon (pentose)and six carbon (hexose) sugars in the presence of high levels ofethanol, limits the efficiency and therefore the economic feasibility oflarge-scale fermentations of certain lignocellulosic carbon sources thatcontain high levels of hemicellulosic biomass. As an example of this, inthe absence of corn, the maize plant is comprised of about 24% Xyloseand 2% arabinose, both of which are pentose sugars that are poorlyutilized by the industrial yeast strains currently employed (Antoni etal., Appl Microbiol Biotechnol 77:23-35 (2007)).

Several groups have attempted to traverse this problem by geneticallyengineering strains of Saccharomyces cerevisiae to contain enzymesnecessary for efficient metabolism of xylose. In initial attempts,various bacterial xylose isomerases were expressed in S. cerevisiae(Amore et al., Appl Microbiol Biotechnol 30:351-357 (1989); Ho et al.,Biotechnol Bioeng Symp 13:245-250 (1983); Moes et al., Biotechnol Lett18:269-274 (1996); Sarthy et al., Appl Environ Microbiol 53:1996-2000(1987); Walfridsson, et al., Appl Environ Microbiol 62:4648-51 (1996)).However, only minimal xylose metabolism was found in these recombinantyeast at temperatures suitable for industrial application.

Other groups have tried to enhance the ethanolic fermentation of S.cerevisiae by exogenously expressing P. stipitis xylose reductase (XR)and xylose dehydrogenase (XDH) (Kötter and Ciriacy, Appl MicrobiolBiotechnol 38:776-783 (1993); Tantirungkij et al., J Ferm Bioeng75:83-88 (1993); Walfridsson et al., Appl Microbiol Biotechnol48:218-224 (1997)). These studies also failed to yield recombinant S.cerevisiae strains that utilized xylose for high yield ethanolicfermentation.

U.S. Pat. No. 7,091,014 to Aristidou et al. describes the geneticengineering of fermenting microorganisms, including S. cerevisiae andSchizosaccharomyces pombe, to express an NAD-dependent glutamatedehydrogenase (GDH) or malic enzyme (ME). These modified yeast displaymodest increases in ethanol and xylitol production, but do not appear tometabolize xylose any faster than control strains lacking the GDH or MEenzymes.

U.S. Pat. No. 7,253,001 to Wahlbom et al. provides geneticallyengineered yeast for the ethanolic fermentation of xylose. Theengineered yeast of U.S. Pat. No. 7,253,001 recombinantly expressexogenous genes for xylose reductase, xylitol dehydrogenase,xylulokinase, phosphoacetyltransferase, aldehyde dehydrogenase, andoptionally phosphoketolase.

Similarly, U.S. Pat. No. 7,226,735 to Jeffries and Jin providesgenetically engineered yeast strains comprising heterologous genesequences encoding xylose reductase, xylitol dehydrogenase, andD-xylulokinase enzymes, which are capable of performing fermentation ofxylose. U.S. Pat. No. 7,285,403 to Jeffries et al. provides similarengineered yeast strains that additionally display reduced PHO13expression.

One drawback to using these genetically engineered yeast strains forfood and beverage production is that the products, such as ethanol andxylitol, may be regulated as novel GMO (genetically modified organism)produced food. Such regulations may result in additional safety andlabeling requirements that are not needed for foods produced by usingunmodified organisms. As such, there remains a need in the art formethods of efficiently fermenting pentose and hexose sugars without theuse of genetically modified organisms.

Xylitol, a five carbon sugar alcohol, is an increasingly utilized sugarsubstitute with several desirable properties. First several studies haveshown that xylitol provides anticariogenic effects that promote oralhealth (Tanzer J M., Int Dent J. 1995 February; 45(1 Suppl 1):65-76).Secondly, xylitol metabolism is not regulated by the insulin pathway,which makes this sweetener an attractive sugar substitute for diabetics.Similarly, xylitol is an appropriate sugar substitute for individualswho suffer from glucose-6-phosphate dehydrogenase deficiencies. Finally,xylitol has fewer calories and net effective carbohydrates than doestable sugar, making it a viable dietary substitute for sucrose.

Although xylitol is present in many fruits and vegetables, extraction isinefficient and uneconomical. As such, xylitol is industrially producedthrough the chemical reduction of xylose. Typically, xylan-containingbiomass is hydrolyzed to produce a mixture of pentose and hexose sugars,including D-xylose. After enrichment, D-xylose is then converted toxylitol in a chemical process using e.g. a nickel catalyst such asRaney-nickel. Many procedures for this process have been developed, forexample see U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, 4,008,285,and 3,586,537. However, the use of xylitol is still limited due to thehigh costs of production and purification. Accordingly, improvedbiotechnological processes for the production of xylitol, especiallyfrom readily available carbon sources such as corn, sugar cane, andvarious wood sources high in hemicellulosic biomass, are highlydesirable.

Several xylose-metabolizing yeast species have been suggested for use inthe production of xylitol, including species of Candida (WO 90/08193, WO91/10740, WO 88/05467, U.S. Pat. No. 5,998,181), mutant and geneticallymodified Kluyvermyces (U.S. Pat. No. 6,271,007), Debaryomyces (Rivas etal., Biotechnol Bioeng. 2008 Oct. 3) and genetically modifiedSaccharomyces (U.S. Pat. No. 7,226,761). However, use of the aboveyeasts have failed to translate into economically viable industrialprocedures for the biotechnological production of xylitol. As such,there remains a need in the art for processes that utilizexylose-metabolizing microorganisms in the industrial production ofxylitol.

Nitrogen fixation refers to the biological process by which atmosphericnitrogen (N₂) is converted into ammonia. This process is essential forlife because fixed nitrogen is required for the biosynthesis of bothamino acids and nucleotides and as such is required for all plantgrowth. Unfortunately, most plants, including industrially andcommercially important crops, are unable to fix nitrogen. These plantsrely on nitrogen fixation from various prokaryotes, termed diazotrophs,including species of bacteria and actinobacteria.

Due to the high fixed nitrogen requirements, fixed nitrogen is commonlya limiting resource for plant growth. To combat this, farmers typicallyrely on fertilizers to supplement the fixed nitrogen content of the soilused for crop growth.

Despite the need for fixed nitrogen supplementation, there are severaldisadvantages to the use of fertilizers and in particular chemicallysynthesized inorganic fertilizers. For example, synthesized nitrogenrequires high levels of fossil fuels such as natural gas and coal, whichare limited resources. In fact, according to the InternationalFertilizer Industry Association (IFA), production of synthetic ammoniacurrently consumes nearly 2% of the world energy production with morethan 100 million metric tons of ammonia being produced in 2008.

In addition, the run-off of nitrogen-rich compounds found in fertilizersis suspected to be a major contributor to the depletion of oxygen inmany parts of the ocean, especially in coastal zones, such as off thecoast of the pacific northwestern region of North America. Similarly,methane and nitrous oxide emissions resulting form the use of ammoniumbased fertilizers may contribute to global climate change, as greenhousegasses.

Practically speaking, the high cost of growing food crops and biomassfor the production of bioenergy (i.e., bioethanol) is in part due to thehigh cost of fertilizers. As such, methods of nitrogen fixation and cropfertilization that reduce or eliminate the reliance on chemicallysynthesized fertilizers are needed to reduce the environmental,agricultural, and financial impact that accompany the use of traditionalfertilizers.

The present invention provides three novel yeast isolates that arecapable of metabolizing a wide range of pentose and hexose sugars, aswell as novel methods for the production of bioethanol and xylitol, thefixation of nitrogen, and crop fertilization, which satisfy these andother needs in the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel endophytic yeaststrains capable of metabolizing both pentose and hexose sugars. In acertain embodiment, the yeast strains are selected from the groupconsisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosastrain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorulamucilaginosa strain Ad1. In specific embodiments, the stains areidentified by an rRNA gene sequence selected from any one of SEQ IDNOS:7 to 18.

In a second aspect, the present invention provides biologically purecultures of the novel endophytic yeast strains of the invention.Cultures of the invention may comprise either a single strain ofendophytic yeast or a mixture of microorganisms comprising at least oneof the novel yeast strains provided herein.

In another aspect of the invention, methods of producing ethanol areprovided. In one embodiment, a method of producing ethanol is providedcomprising fermenting a carbon source with an endophytic strain of yeastthat is capable of metabolizing both pentose and hexose sugars. Incertain embodiments, the endophytic strain is selected from the groupconsisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosastrain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorulamucilaginosa strain Ad1. In other embodiments, the endophytic strain isidentified by an rRNA gene sequence selected from the group consistingof SEQ ID NOS:7 to 18.

In yet another aspect of the invention, methods of producing xylitol areprovided. In one embodiment, the method comprises fermenting a carbonsource with an endophytic strain of yeast capable of metabolizing bothpentose and hexose sugars. In certain embodiments, the endophytic strainis selected from the group consisting of Rhodotorula graminis strainWP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosastrain PTD3, and Rhodotorula mucilaginosa strain Ad1. In otherembodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18.

In one aspect of the invention, methods of producing mixtures of xylitoland ethanol are provided. In one embodiment, the method comprisesfermenting a carbon source with an endophytic strain of yeast capable ofmetabolizing both pentose and hexose sugars. In certain embodiments, theendophytic strain is selected from the group consisting of Rhodotorulagraminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorulamucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. Inother embodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18.

In another aspect, the present invention provides methods of producingsubstantially pure ethanol and/or xylitol. In certain embodiments, themethods comprise the steps of producing a mixture of xylitol and ethanoland purifying said xylitol and ethanol from the residual material. Inone embodiment, the method comprises fermenting a carbon source with anendophytic strain of yeast capable of metabolizing both pentose andhexose sugars. In certain embodiments, the endophytic strain is selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, theendophytic strain is identified by an rRNA gene sequence selected fromthe group consisting of SEQ ID NOS:7 to 18.

In yet another aspect, the invention provides a method of producingxylitol, the method comprising the steps of hydrolytically treating asource of biomass, separating a first stream comprising xylose from asecond stream comprising glucose, and fermenting said first stream withan endophytic strain of yeast capable of metabolizing both pentose andhexose sugars. In certain embodiments, the endophytic strain is selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, theendophytic strain is identified by an rRNA gene sequence selected fromthe group consisting of SEQ ID NOS:7 to 18.

In certain embodiments, the method further comprises fermenting saidsecond stream with a yeast capable of producing ethanol. In a particularembodiment, the yeast is an endophytic strain capable of metabolizingboth pentose and hexose sugars. In certain embodiments, the endophyticstrain is selected from the group consisting of Rhodotorula graminisstrain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorulamucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. Inother embodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18.

In one aspect, the invention provides methods of producing animalfeedstock. In certain embodiments, the methods comprise fermenting acarbon source. In certain embodiments, the endophytic strain is selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, theendophytic strain is identified by an rRNA gene sequence selected fromthe group consisting of SEQ ID NOS:7 to 18.

In another aspect, the invention provides a recombinant yeast capablefermenting both hexose and pentose sugars. In certain embodiments, theyeast harbors a heterologous gene sequence from an endophytic yeaststrain. In certain embodiments, the endophytic strain is selected fromthe group consisting of Rhodotorula graminis strain WP1, Rhodotorulamucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, andRhodotorula mucilaginosa strain Ad1. In other embodiments, theendophytic strain is identified by an rRNA gene sequence selected fromthe group consisting of SEQ ID NOS:7 to 18. In certain embodiments, therecombinant yeast is a Saccharomyces or a Schizosaccharomyces yeaststrain.

In yet another aspect, the invention provides a method of producingethanol. In one embodiment, the method comprises fermenting a carbonsource with a recombinant yeast capable of fermenting both pentose andhexose sugars. In certain embodiments, the yeast comprises aheterologous gene sequence from an endophytic yeast strain capable offermenting both pentose and hexose sugars. In certain embodiments, theyeast comprises a heterologous gene sequence from an endophytic yeaststrain capable of fermenting both pentose and hexose sugars. In certainembodiments, the endophytic strain is selected from the group consistingof Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strainPTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosastrain Ad1. In other embodiments, the endophytic strain is identified byan rRNA gene sequence selected from the group consisting of SEQ ID NOS:7to 18. In certain embodiments, the recombinant yeast is a Saccharomycesor a Schizosaccharomyces yeast strain.

In one aspect, the invention provides novel Xylose Dehydrogenase (XDH)and Xylose Reductase (XR) genes and coding sequences cloned from theendophytic yeast provided herein, as well as the polypeptides encodedtherein.

In another aspect, the invention provides a method of fixing nitrogencomprising the use an endophytic yeast of the invention or a recombinantorganism harboring a heterologous gene from an endophytic yeast providedherein. In certain embodiments, the method comprises fertilization of acrop.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Photomicrographs of the yeast strains (scale bar equal to 25 mmfor A and 10 mm for B, C, D). (A) WP1; (B) PTD2; (C) PTD3; (D)Rhodotorula glutinis.

FIG. 2. Phylogenetic tree showing relatedness among 18 S gene sequencesof yeast strains. The tree was constructed with a total of 952 positionsusing a neighbor joining distance matrix. Evolutionary distances werecomputed using the Jukes-Cantor method. Bootstrap values (1000 treeinteractions) are indicated at the nodes.

FIG. 3. Phylogenetic reconstruction based on ITS1-5.8S-ITS2 sequences ofyeast strains. The tree was constructed with a total of 583 positionsusing a neighbor-joining distance matrix. Evolutionary distances werecomputed using the Jukes-Cantor method. Bootstrap values (1000 treeinteractions) are indicated at the nodes.

FIG. 4. Phylogenetic tree showing relatedness among large subunit geneD1/D2 region sequences of yeast strains. The tree was constructed with atotal of 586 positions using a neighbor-joining distance matrix.Evolutionary distances were computed using the Jukes-Cantor method.Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 5. Phylogenetic tree showing relatedness among 18 S gene sequencesof yeast strains. The tree was constructed with a total of 952 positionsusing a Maximum Parsimony method. Bootstrap values (1000 treeinteractions) are indicated at the nodes.

FIG. 6. Phylogenetic reconstruction based on ITS1-5.8S-ITS2 sequences ofyeast strains. The tree was constructed with a total of 583 positionsusing a Maximum Parsimony. Bootstrap values (1000 tree interactions) areindicated at the nodes.

FIG. 7. Phylogenetic tree showing relatedness among large subunit geneD1/D2 region sequences of yeast strains. The tree was constructed with atotal of 586 positions using a Maximum Parsimony method. Bootstrapvalues (1000 tree interactions) are indicated at the nodes.

FIG. 8. Clustering of phenotypic characteristics profiles of the studiedyeast strains (WP1, PTD2, PTD3, ATCC, and Baker's yeast) and thereference species of the API 20C AUX system (bioMerieux, 2007) based onthe overall similarity. The distance between any two clusters wasdetermined by the Ward's minimum variance method (Milligan, 1980).

FIG. 9. IAA production by yeast strains incubated with 0.1%L-tryptophan.

FIG. 10. Growth rates of the yeast strains WP1, PTD2, PTD3, ATCC, andBaker's yeast in rich medium.

FIG. 11. Experimental growth curves for the yeast strains WP1, PTD2,PTD3, ATCC, and Baker's yeast in YPG medium.

FIG. 12. Glucose consumption, glycerol production, and ethanolproduction in a culture PTD3 grown in glucose.

FIG. 13. HPLC chromatograms for the data provided in FIG. 15, showingthe amount of the xylose peak at the beginning of the experiment.

FIG. 14. HPLC chromatograms for the data provided in FIG. 15, showingthe reduction of the xylose peak and formation of the xylitol peak atthe end of the experiment.

FIG. 15. Consumption of glucose and xylose and production of xylitol andethanol in a culture of PTD3 yeast grown in glucose and xyloseseparately.

FIG. 16. Conversion of xylose to xylitol by the yeast strains WP1, PTD2,and PTD3 cultured in xylose.

FIG. 17. Consumption of glucose and xylose and production of xylitol andethanol in a culture of WP1 yeast grown in glucose and xyloseseparately.

FIG. 18. Consumption of glucose and xylose and production of xylitol andethanol in a culture of PTD2 yeast grown in glucose and xyloseseparately.

FIG. 19. Consumption of glucose and xylose and production of xylitol andethanol in a culture of PTD3 yeast grown in glucose and xyloseseparately.

FIG. 20. Effect of medium conditions on the consumption of glucose andthe production of ethanol and xylitol by PTD3 yeast.

FIG. 21. Effect of medium conditions on the consumption of xylose andthe production of ethanol and xylitol by PTD3 yeast.

FIG. 22. Effect of yeast concentration on the consumption of glucose andthe production of ethanol and xylitol by PTD3 yeast.

FIG. 23. Effect of yeast concentration on the consumption of xylose andthe production of ethanol and xylitol by PTD3 yeast.

FIG. 24. Fermentation of glucose to ethanol by PTD3 yeast cultured inglucose with MS and yeast extract.

FIG. 25. Fermentation of xylose to xylitol by PTD3 yeast cultured inxylose with MS and yeast extract.

FIG. 26. Double fermentation of glucose and xylitol to ethanol andxylitol by PTD3 yeast cultured in glucose and xylose together and MS andyeast extract.

FIG. 27. Growth rate of PTD3 yeast in medium containing glucose andxylose.

FIG. 28. Mixed fermentation of hexose (28A) and pentose (28B) sugars byPTD3 yeast cultured in MS and yeast extract with arabinose, galactose,glucose, xylose and mannose.

FIG. 29. Growth rate of PTD3 yeast grown in mediums containing mixedsugars, arabinose, xylose, glucose, galactose, and mannose with MS andyeast extract.

FIG. 30. Growth rate of PTD3 yeast grown in mediums containing mixedsugars, arabinose, xylose, glucose, galactose, and mannose with MS andyeast extract.

FIG. 31. Mixed fermentation of glucose and xylitol to ethanol andxylitol by PTD3 yeast cultured in arabinose, xylose, glucose, galactose,and mannose with MS and yeast extract.

FIG. 32. 18 S Ribosomal sequence (SEQ ID NO:18) for the Ad1 yeastisolated from Arundo donax (giant reed).

FIG. 33. Growth curve of Rhodotorula graminis strain WP1 andSaccharomyces cerevisiae (ATCC strain #6037) in nitrogen-free MS medium(Caisson) containing dextrose and mannitol. Growth in three flasks ofeach strain was monitored for 3 days. The experiment was repeated withsimilar results.

FIG. 34. (A) Growth of WP1 and PTD3 in MS medium with 3% glucose as thecarbon source. Baker's yeast (BK; ATCC6037) was used as a positivecontrol. The experiments were performed in triplicate and the error barsindicate the standard deviations. (B) Growth of WP1 and PTD3 in MSmedium with 3% xylose as the carbon source. Baker's yeast (BK) was usedas a control. The experiments were performed in triplicate and the errorbars indicate the standard deviations.

FIG. 35. Exon/Intron structures of the XR and XDH-encoding genes ofRhodotorula graminis strain WP1.

FIG. 36. Exon/Intron structures of the XR and XDH-encoding genes ofPichia stipitis.

FIG. 37. Amplification of WP1 XR mRNA (A) and XDH mRNA (B) from cellsgrown in glucose or xylose. The first lane is a Fermentas 1 kb DNAladder. RNA templates directly subjected to a regular PCR (withoutreverse transcriptase) served as negative controls for both genes. The 1kb bands were cloned and the sequences were verified to be XR andXDH-encoding genes.

FIG. 38. Amplification of PTD3 XDH mRNA and XR mRNA from cells grown inglucose (“glu”) or xylose (“xyl”). The first lane is a Fermentas 100 bpDNA ladder.

FIG. 39. WP1 and PTD3 XR/XDH gene expressions from cells grown in 2%glucose (lanes 1, 4, 7, 10), 1% glucose+1% xylose (lane 2, 5, 8, 11),and 2% xylose (lanes 3, 6, 9, 12). Lane 1-6: WP1 gene expression; Lane7-12: PTD3 gene expression.

FIG. 40. WP1 and PTD3 XR (right)/XDH (left) gene expressions from cellsgrown in 2% glucose, 2% xylose and 2% glucose+2% xylose. RNA templatesused in different conditions were labeled in the figure.

FIG. 41. WP1 and PTD3 XR/XDH gene 18 S rRNA RT-PCR from cells grown in2% glucose (lanes 1, 3), 2% glucose+2% xylose (lanes 2, 4) and 2% xylose(lanes 3, 6) medium. Lane S is a Fermentas 1 kb DNA ladder.

FIG. 42. Corn growth after 11 weeks in nitrogen-limited conditions withor without WP1 inoculation. Three different corn varieties (lines 1, 2,and 3) were planted in each container. Biomass of the uninoculatedplants was 9.3 g, 3.9 g, and 15.0 g whereas the biomass of the WP1inoculated plants was 63 g, 87.1 g, and 45.1 g. In addition, the %viability in WP1 colonized plants (58-92%) was higher than uninoculatedplants (8.3-29.2%) plants. Statistical analysis indicated significantdifferences (P≦0.1) for both viability and biomass with WP1 symbioticplants having higher viability and biomass compared to uninoculatedplants.

FIG. 43. Greenhouse studies with corn (line-3) that were eitherNS=nonsymbiotic (uninoculated control) or symbiotic (N=9) withRhodotorula sp. WP1 in the absence of stress. The biomass, yields andheights of plants were assessed, and WP1 colonized plants were found tobe larger and produced higher yields (ears) than NS plants. (P≦0.004).N=9; SE≦0.21, 0.56, and 0.27 for biomass, yields, and heights,respectively.

FIG. 44. PTD3 XDH-encoding gene open reading frame (SEQ ID NO:47).

FIG. 45. PTD3 XR-encoding gene open reading frame (SEQ ID NO:45).

FIG. 46. WP1 XR-encoding gene open reading frame (SEQ ID NO:41).

FIG. 47. WP1 XDH-encoding gene open reading frame (SEQ ID NO:43).

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

In one aspect, the present invention provides novel endophytic yeaststrains, including WP1 and PTD3, isolated from within the stems ofpoplar (Populus) trees, which were genetically characterized withrespect to their xylose metabolism genes. These strains, belonging tospecies Rhodotorula graminis and R. mucilaginosa, respectively, utilizeboth hexose and pentose sugars, including the common plant pentosesugar, D-xylose. In another aspect, the present invention provides thexylose reductase gene (XYL1) and xylitol dehydrogenase gene (XYL2) fromthese yeast strains, which were cloned and characterized. The derivedamino acid sequences of xylose reductase (XR) and xylose dehydrogenase(XDH) are 32°˜41% homologous to those of Pichia stipitis and Candida.Spp., species known to utilize xylose. The derived XR and XDH sequencesof WP1 and PTD3 have higher homology (73% and 69% identity) with eachother. WP1 and PTD3 were grown in single sugar and mixed sugar medium toanalyze the XYL1 and XYL2 gene regulation mechanisms. These resultsrevealed that for both strains, the gene expression is induced byD-xylose, and that the expression was not repressed by glucose in thepresence of xylose in PTD3.

Notably, the gene expression of the WP1 and PTD3 is unique in theseendophytic yeast strains. They are expressed in response to xylose evenwhen glucose is present. In contrast, in other species, these xylosemetabolism genes are shut off when glucose is present, preventingsimultaneous use of both 5-carbon and 6-carbon sugars.

Lignocellulosic material containing cellulose, hemicellulose, and ligninis an abundant renewable organic resource that can be used for theproduction of energy and biochemicals. The conversion of both thecellulose and hemicellulose fractions for production of biochemicals isbeing studied intensively. Between 23% to 40% of the lignocellulosicbiomass consists of hemicellulose, the main component being xylose inmost hardwoods and annual plants (Lee et al. 1979). Whereas thefermentation of glucose can be carried out efficiently by the commonbrewer's yeast (Saccharomyces cerevisiae), the bioconversion of thepentose fraction (xylose and arabinose) presents a challenge since it isnot metabolized by this species. In the past decades, numerous studieshave been carried out on various aspects of D-xylose bioconversion (DuPreez 1994; Winkelhausen and Kuzmanova 1998).

D-xylose can be utilized by bacteria, yeasts and fungi (Jeffries 1983)using different pathways. In one pathway, D-xylose can be directlyconverted to D-xylulose by xylose isomerase (Aristidou and Penttila2000) without the participation of cofactors. In some yeasts and fungi,conversion of D-xylose to D-xylulose is carried out more often by twoenzymatic steps. First, D-xylose is reduced by a NADPH/NADH-linkedxylose reductase (XR) to xylitol, followed by oxidation of xylitol toxylulose by an NAD-linked xylitol dehydrogenase (XDH) (Bruinenberg andvan Dijken 1983). D-xylulose is subsequently phosphorylated toD-xylulose-5-phosphate by D-xylulokinase before it enters the pentosephosphate, Embden-Meyerhof, and phosphoketolase pathways (Skoog andHahn-Hagerdal 1988).

The two major chemicals of interest that can be produced from D-xyloseby yeasts are ethanol and xylitol. It is known that under normal growthconditions, some pentose-fermenting yeasts (e.g. Pichia stipitis)produce mostly ethanol (Du Preez 1994; Schneider 1989); while others(e.g. Candida guilliermondii, Candida tropicalis) produce mainly xylitolas the end products (Barbosa et al. 1988; Gong et al. 1981). As anintermediate metabolite, xylitol is widely applied in food andpharmaceutical industries because of its equivalent sweetness to sucroseand high negative heat of solution (Borges 1991; Passon 1993), itsanti-cariogenic and anti-infection effects (Pizzo et al. 2000; Sakai etal. 1996; Brown et al. 2004), and independent metabolism of insulin,therefore making it useful for diabetic patients (Salminen et al. 1989).Among the xylose-fermenting yeast, the genus Candida is one of the mostefficient xylitol producers (Meyrial et al. 1991). Ojama demonstratedthat C. guilliermondii VTT-C-71006 is an efficient xylitol producer. Axylitol yield of 0.74g/g xylose was obtained within 50 hours at aninitial D-xylose concentration of 100g/1 (Ojama 1994).

The pink yeast strains WP1 (Rhodotorula graminis) and PTD3 (Rhodotorulamucilaginosa) provided in one aspect of the present invention areremarkable for their good performance in xylitol production(approximately 67% conversion) and sugar metabolism in the presence ofseveral common fermentation inhibitors (Vajzovic, A., unpublished). Sofar, investigation of xylitol production by yeasts has been limited toCandida and Pichia species and studies of D-xylose metabolism inRhodotorula spp. were barely reported. Although XR and XDH activitieswere detected in Rhodosporidium toruloides (the teleomorph ofRhodotorula glutinis)(Freer et al. 1997), none of the genes encoding XRand XDH were cloned from the Rhodotorula genus. The present inventionprovides, among other aspects, the first report that describes thecloning and characterization of the XR-encoding gene (XYL1) andXDH-encoding gene (XYL2) from both Rhodotorula graminis and Rhodotorulamucilaginosa yeast strains.

In one aspect, the present invention provides XR and XDH encoding genes,which were cloned from Rhodotorula graminis strain WP1. The expressionof the two genes was verified by RT-PCR. This study shows that D-xyloseis a good inducer of XR and XDH in both strains. This is similar to thetrend found with Candida guilliermondii (Sugai and Delgenes 1995) andPichia stipitis (Bichio et al. 1988). Furthermore, a novelcharacteristic of lack of inhibition by glucose for these genes is alsodemonstrated.

Notably, the XI (xylose isomerase)-encoding gene was not found in theWP1 genome sequence provided by the JGI. Thus WP1 likely utilizes thetwo-step redox pathway in D-xylose metabolism as in other yeasts.However, the alignments showed that the XR and XDH sequences have lowhomology (32% ˜41% identities) with other XRs and XDHs from Candida spp.and Pichia stipitis yeasts. In addition, the WP1 XR and XDH-encodinggenes have multiple introns and the exon/ intron structures are morecomplicated and advanced than the homologous genes in Pichia stipitisand Candida spp. These differences might introduce greater variabilityof protein sequences translated from a single gene and might have animpact on enhancing the expression of the XR and XDH genes (Smith andLee 2008; Lin et al. 2010). From the macro perspective, the genedifferences suggest that there could be long evolution distances betweenWP1 and Pichia stipitis and Candida spp. and this might lead to someother differences in the xylose metabolism pathway between these yeasts.

The present study of gene expression levels in xylose and glucose mediashows that the expression of WP1 XR and XDH-encoding genes were inducedby xylose. The two genes in WP1 were expressed to low levels while grownin glucose medium. Additionally, the expression level of theXDH-encoding gene (XYL1) in WP1 was higher than that of the XR-encodinggene (XYL2).

In a related aspect, the present invention also provides full-length XRand XDH encoding genes, which were cloned from Rhodotorula mucilaginosastrain PTD3. The expression of these two genes was also verified byRT-PCR. Sequence alignment results show that the XR and XDH sequencesalso have low homology (37%˜41% identities) with other XRs and XDHs fromCandida spp. and Pichia stipitis yeasts. Since the genome sequence ofPTD3 is not available, the exon/intron structures of the two genes wasnot determined. However, based on the high homology (73% and 69%identity for XR, XDH) with WP1, it is likely that the gene structures ofPTD3 may be more like that of WP1 and that these two endophytic yeastsof poplar trees may metabolize D-xylose using the same pathway.

Like in WP1, the expression of PTD3 XR and XDH-encoding genes was alsoinduced by xylose. The two genes in PTD3 were expressed to low levelswhile grown in glucose medium. As in WP1, the expression level of theXDH-encoding gene (XYL1) in PTD3 was higher than that of the XR-encodinggene (XYL2).

Since PTD3 grew better in D-xylose medium compared to WP1, onehypothesis to explain this difference is that PTD3 may produce theenzymes involved in xylose metabolism at higher levels than does WP1.This gene expression study verified that both the XR and XDH-encodinggene expression levels were much higher in PTD3 than in WP1, thussupporting this hypothesis. Further study into the resulting proteinlevels and also the xylose uptake mechanisms for these yeast strains isyet to be explored.

In another aspect of the invention, single sugars and mixed sugars wereinvestigated to analyze their potential to induce XR and XDH-encodinggene expressions in both WP1 and PTD3. For many yeasts likeSaccharomyces cerevisiae, Pichia stipitis and Candida spp., D-glucose isthe preferred substrate for growth and fermentation when both D-glucoseand D-xylose are present in the medium. The genes for xyloseassimilation (XYL1, XYL2) were not expressed in Pichia stipitis in thepresence of glucose in the medium (Jeffries et al. 2007). The presentstudy shows that in both WP1 and PTD3 yeast strains, the two genes werestill expressed in response to xylose in the presence of glucose in themedium. Furthermore, the band quantities of RT-PCR in single sugar(xylose) and mixed sugar (glucose +xylose) revealed that the two geneswere not repressed by glucose in PTD3 while they were slightlysuppressed in WP1. These are significant results because xylosereductase and xylitol dehydrogenase are pivotal for growth and xylitolformation during xylose metabolism. And the high-level expression ofboth genes in the mixed sugars of xylose and glucose will largelyincrease the xylitol yield in mixed sugars from real hydrolytes and willcontribute to optimizing fermentation conditions of lignocellulosicbiomass. In addition, better understanding of the regulation mechanismof these genes, together with identification of the XR and XDH-encodinggenes as well as the xylose uptake genes will help determine thestrategies for genetic engineering of industry strains such asS.cerevisiae for further improvement of productivity. Accordingly, thepresent invention provides, in one aspect, recombinant yeast cells andstrains harboring a heterologous XR and/or XDH-encoding gene from theWP1 or PTD3 strain, or a highly similar sequence.

In one aspect, the present invention provides a biotechnological processfor the production of a sugar alcohol or polyol, using the endophyticyeast provided herein. One novel yeast strain, provided herein, wasisolated from poplar trees and has several unique properties. In oneembodiment, the invention provides a PTD3 yeast strain isolated from ahybrid poplar tree or a giant reed.

Pretreatment of lignocellulosic biomass can produce fermentationinhibitors such as furfural, 5-HMF, and acetic acid. These compounds candecrease the ethanol yields from sugars. Provided herein are isolatedyeast strains that have a high tolerance for such inhibitors. Asystematic study of the effect of furfural, 5-HMF, and acetic acidconcentration on the fermentation of glucose and xylose to ethanol andxylitol respectively by PTD3, a novel, genetically unmodified yeast isprovided herein.

The influence of furfural in different concentrations (from 1 to 5 g/L)on the growth of PTD3 yeast under cultivation in synthetic nutrientmedia has been studied. The yeast provided herein grow well in presenceof furfural and showed resemblance in growth and fermentative patternwith controls. Ethanol yield achieved from glucose and xylitol, usingthe yeast strains of the invention, were of 90% of theoretical yield forethanol and 70% of the theoretical yield for xylitol. Ethanol yieldsfrom glucose were not influenced by presence of furfural. However,xylitol biosynthesis was affected by the presence of furfural in thefermentation media. The effects of higher concentrations of furfural (10and 20 g/L) on the ethanol and xylitol yields are presented herein, aswell as the effects of 5-HMF and acetic acid.

Up to date, there is no reported microorganism that is capable ofutilizing both, hexose and pentose sugars at the same time, withoutbeing genetically modified or co-cultured. A genetically unmodifiedyeast which is capable of rapid assimilation and catabolism of five andsix carbon sugars (arabinose, xylose, galactose, glucose and mannose) isprovided herein. This yeast (PTD3) was shown not to be subject tohexose-mediated repression during mixed sugars fermentation. PTD3produced ethanol of 82% of theoretical during fermentation of glucose,mannose and galactose. It produced considerable amount of xylitol of96.1% of theoretical when xylose was present in the fermentation media.The high ethanol and xylitol were obtained without media, aeration,temperature and pH optimization.

The novel yeast provided herein also have a high tolerance ofinhibitors, including without limitation, furfurals, 5-HMF, and aceticacid, during biological production of ethanol and xylitol. PTD3 caneffectively ferment five and six carbon sugars present in hydrolysatesfrom different cellulosic biomass, for example, steam pretreatedswitchgrass, hybrid poplar, and sugar cane bagasse, to ethanol andxylitol.

II. Endophytic Yeast Strains and Cultures Thereof

In one embodiment of the invention, novel endophytic yeast strainscapable of metabolizing both pentose and hexose sugars are provided. Incertain embodiments, these yeast strains are most closely related toRhodotorula graminis or Rhodotorula mucilaginosa species. In aparticular embodiment, the novel strains of the invention are selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In one embodiment of theinvention, the novel endophytic yeast strains contain an rRNA genesequence that is selected from any one of SEQ ID NOS:7 to 18. In aparticular embodiment, an endophytic yeast strain of the invention mayhave an 18 S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In another embodiment, the present invention provides cultures of novelendophytic yeast strains capable of metabolizing both pentose and hexosesugars. In some embodiments, the cultures of the invention comprise abiologically pure culture of an endophytic yeast strain, while in otherembodiments, the cultures of the invention may comprise more than onestrain of yeast. In specific embodiments, the cultures of the inventionmay comprise a yeast strain selected from the group consisting ofRhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2,Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosastrain Ad1. In other embodiments, the cultures of the invention compriseone or more yeast strain that is identified by an rRNA gene sequenceselected from the group consisting of SEQ ID NOS:7 to 18.

The novel endophytic yeast strains and cultures of the present inventionmay be useful in the fermentative production of bioethanol, xylitol, andother biotechnological manufacturing products. In a particularembodiment, the novel strains of the invention are useful for thefermentation of mixtures of pentose and hexose sugars. In certainembodiments, the strains and cultures of the invention are useful forthe fermentation of biomass that has been pretreated to yield a mixtureof pentose and hexose sugars. For example, lignocellulosic biomass suchas wood or wood residuals (e.g., saw mill or paper mill discards),municipal paper waste (e.g., newspapers), agricultural residuals (e.g.,corn stover, sugarcane bagasse), tall woody grasses, and the like.Methods of pretreating lignocellulosic biomass for yeast fermentationare well known in the art and include both acid hydrolysis and enzymatichydrolysis. For review, see Lange J. P., Biofuels, Bioproducts, andBiorefining 1(1):39-48 (2007); Jørgensen H. et al., Biofuels,Bioproducts, and Biorefining 1(2):119-134 (2007); Wyman C. E. et al.,Bioresour Technol. 2005 December; 96(18):2026-32; and Wyman C. E. etal., Bioresour Technol. 2005 December; 96(18):1959-66.

In another embodiment, the endophytic yeast strains and cultures of thepresent invention may be useful for fixing atmospheric nitrogen. In aparticular embodiment, the novel strains of the invention are useful forfertilizing a crop in the presence or absence of a traditional chemicalfertilizer. In one embodiment, the novel strains are useful forinoculating a crop or colonizing the soil a crop is planted in with theyeast. The soil may be colonized with the yeast prior to planting thecrop, for example before, during, or after tilling the soil inpreparation for planning the crop. In other embodiments, the soil may becolonized with the yeast after the crop has been planted.

In some embodiments, the cultures of the invention useful for nitrogenfixation and/or fertilization of a crop comprise a biologically pureculture of an endophytic yeast strain, while in other embodiments, thecultures of the invention may comprise more than one strain of yeast. Inspecific embodiments, the cultures of the invention may comprise a yeaststrain selected from the group consisting of Rhodotorula graminis strainWP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosastrain PTD3, and Rhodotorula mucilaginosa strain Ad1. In otherembodiments, the cultures of the invention comprise one or more yeaststrain that is identified by an rRNA gene sequence selected from thegroup consisting of SEQ ID NOS:7 to 18.

III. Methods for Producing Ethanol, Sugar Alcohols, and Polyols

In one embodiment, the present invention provides novel methods forproducing ethanol comprising fermenting a carbon source with anendophytic strain of yeast. In certain embodiments, the endophytic yeastis capable of metabolizing both pentose and hexose sugars. In a specificembodiment, the endophytic strain is selected from the group consistingof Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strainPTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosastrain Ad1. In other embodiments, the endophytic strain is identified byan rRNA gene sequence selected from the group consisting of SEQ ID NOS:7to 18. In a particular embodiment, an endophytic yeast strain of theinvention may have an 18 S rRNA gene sequence selected from SEQ ID NOS:7to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to15.

In another embodiment of the invention, methods of producing xylitol areprovided. In one embodiment, the method comprises fermenting a carbonsource with an endophytic strain of yeast capable of metabolizing bothpentose and hexose sugars. In certain embodiments, the endophytic strainis selected from the group consisting of Rhodotorula graminis strainWP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosastrain PTD3, and Rhodotorula mucilaginosa strain Ad1. In otherembodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18. In aparticular embodiment, an endophytic yeast strain of the invention mayhave an 18 S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

The carbon sources used in the methods of the invention, may comprise apentose sugar or sugar alcohol, a hexose sugar or sugar alcohol, or acombination thereof. In particular embodiments, the carbon source isselected from the group consisting of glucose, glycerol, calcium2-keto-gluconate, arabinose, xylose, adonitol, xylitol, galactose,inositol, sorbitol, methyl-α-glucopyranoside, N-acetyl-glucosamine,cellobiose, lactose, maltose, sucrose, trehalose, melezitose, raffinose,and combinations thereof. In a particular embodiment, the carbon sourceis xylitol, glucose, or a combination of sugars containing xylitol,glucose, or both. In other embodiments, the carbon source may comprisesbiomass that has been hydrolytically pre-treated. For example,lignocellulosic biomass such as wood or wood residuals (saw mill orpaper mill discards), municipal paper waste, agricultural residuals(corn stover, sugarcane bagasse), tall woody grasses, and the like. Thecarbon sources used in the methods of the invention are not limited tothose listed above.

In related embodiments, the present invention provides methods ofproducing mixtures of xylitol and ethanol. In one embodiment, the methodcomprises fermenting a carbon source with an endophytic strain of yeastcapable of metabolizing both pentose and hexose sugars. In certainembodiments, the endophytic strain is selected from the group consistingof Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strainPTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosastrain Ad1. In other embodiments, the endophytic strain is identified byan rRNA gene sequence selected from the group consisting of SEQ ID NOS:7to 18. In a particular embodiment, an endophytic yeast strain of theinvention may have an 18 S rRNA gene sequence selected from SEQ ID NOS:7to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to15. In one particular embodiment, the present invention provides methodsof producing mixtures of xylitol and ethanol.

In certain embodiments, the methods of the invention further comprisepurifying one or more of xylitol, ethanol, or both after production.Methods of purifying xylitol from reaction mixtures are well known inthe art and include, without limitation, distillation, crystallization,chromatography, combinations thereof, and the like. For example, U.S.Pat. No. 6,538,133 describes chromatographic procedures of purifyingxylitol from cultures of xylitol-producing microorganisms. Rivas et al.,J. Agric. Food Chem. 2006, 54(12):4430-4435, describe a process ofpurifying xylitol obtained by fermentation of corncob hydrolysates bycrystallization. Methods of distilling ethanol are also well known inthe art. For example, U.S. Pat. No. 7,297,236 describe processarrangements for distilling fuel grade ethanol. Methods ofsimultaneously producing xylitol and ethanol are well known in the art,for example see U.S. Pat. No. 7,109,055.

In certain embodiments, the methods of the present invention comprisethe steps of producing a mixture of xylitol and ethanol and purifyingsaid ethanol and xylitol from the residual material. In one particularembodiment, the mixture of xylitol and ethanol is first distilled toyield substantially pure ethanol and then xylitol is purified from thedistillation residuals. In one embodiment, the method comprisesfermenting a carbon source with an endophytic strain of yeast capable ofmetabolizing both pentose and hexose sugars. In certain embodiments, theendophytic strain is selected from the group consisting of Rhodotorulagraminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorulamucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. Inother embodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18. In aparticular embodiment, an endophytic yeast strain of the invention mayhave an 18 S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In yet other embodiments of the invention, methods are provided for theproduction of xylitol comprising the steps of hydrolytically treating asource of biomass to produce a mixture of pentose and hexose sugars,separating a first stream comprising xylose from a second streamcomprising glucose, and fermenting said first stream with an endophyticstrain of yeast capable of metabolizing both pentose and hexose sugars.In certain embodiments, the endophytic strain is selected from the groupconsisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosastrain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorulamucilaginosa strain Ad1. In other embodiments, the endophytic strain isidentified by an rRNA gene sequence selected from the group consistingof SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeaststrain of the invention may have an 18 S rRNA gene sequence selectedfrom SEQ ID NOS:7 to 9 or 15 to 16, an ITS rRNA gene sequence selectedfrom SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selectedfrom SEQ ID NOS:13 to 15.

In some embodiments, the above method further comprises fermenting saidsecond stream with a yeast capable of producing ethanol. In a particularembodiment, the yeast is an endophytic strain capable of metabolizingboth pentose and hexose sugars. In certain embodiments, the endophyticstrain is selected from the group consisting of Rhodotorula graminisstrain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorulamucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. Inother embodiments, the endophytic strain is identified by an rRNA genesequence selected from the group consisting of SEQ ID NOS:7 to 18. In aparticular embodiment, an endophytic yeast strain of the invention mayhave an 18 S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15. In someembodiments, the yeast strain used to ferment said first stream is thesame as the yeast strain used to ferment said second stream. In yetother embodiments, the yeast strains are different.

IV. Recombinant Yeast Strains and Methods of Use Thereof

In another aspect, the invention provides recombinant yeast strainscapable of fermenting both pentose and hexose sugars. In certainembodiments, these strains harbor a heterologous gene sequence from anendophytic yeast strain selected from the group consisting ofRhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2,Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosastrain Ad1, wherein said strain is identified by an rRNA gene sequenceselected from any one of SEQ ID NOS:7 to 18.

In one embodiment, the heterologous gene sequence encodes for a xylosereductase (XR) protein or a xylose dehydrogenase (XDH) protein. Incertain embodiments, the heterologous gene sequence has at least 85%sequence identity with an XYL1 or XYL2 gene sequence or coding sequencefrom an endophytic yeast provided herein. In certain embodiments, theheterologous gene sequence has at least 85% sequence identity to anucleotide sequence selected from the group consisting of SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47. In other embodiments, theheterologous gene sequence may have at least about 85% identity, or atleast about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher sequence identity to an XYL1 or XYL2 gene sequence orcoding sequence provided herein. In certain embodiments, theheterologous gene sequence may further comprise one or more introns.

In one embodiment, the heterologous gene sequence encodes for a xylosereductase (XR) protein or a xylose dehydrogenase (XDH) protein. Incertain embodiments, the xylose reductase protein is from the WP1 or thePTD3 stain. In a particular embodiment, the heterologous gene sequenceencodes for a polypeptide having at least about 85%, or at least about86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orhigher sequence identity to an amino acid sequence selected form SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.

In certain embodiments, the heterologous gene sequence may be clonedinto a microbial genome, for example a bacterial or yeast chromosome, ormay comprise an expression vector, a recombinant or artificial microbialchromosome, for example a bacterial (BAC) or yeast (YAC) chromosome, abacterial plasmid, a yeast plasmid, a recombinant bacteria phage, arecombinant viral vector, a mammalian expression vector, a baculovirusvector. In yet other embodiments, the heterologous gene sequence mayencode for a fusion protein. In another embodiment, the heterologousgene sequence may encode for a tagged protein, such as a tagged XR orXDH protein.

In certain embodiments, the recombinant yeast strains may be aSaccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, aBrettanomyces, a Torulaspora, an Ascobotryozyma, a Citeromyces, aDebaryomyces, an Eremothecium, a Issatchenkia, a Kazachstania, aKluyveromyces, a Kodamaea, a Kregervanrija, a Kuraishia, a Lachancea, aLodderomyces, a Nakaseomyces, a Pachysolen, a Pichia, a Saturnispora, aTetrapisispora, a Torulaspora, a Vanderwaltozyma, a Williopsis, and thelike. In a particular embodiment, the recombinant yeast is aSaccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, ora Brettanomyces. In one embodiment, the strain is Saccharomycescerevisiae. In a related aspect, biologically pure cultures of therecombinant yeast strains are provided.

In another aspect, the invention provides methods of fermenting a carbonsource with a recombinant yeast strain provided herein. In certainembodiments, the methods comprise culturing a recombinant yeast of theinvention in the absence of a supplemental nitrogen source, for exampleammonium or nitrate.

In a related aspect, methods of producing ethanol are provided. Incertain embodiments, the methods comprise fermenting a carbon sourcewith a recombinant yeast strain provided herein. In certain embodiments,the methods comprise fermenting a carbon source in the absence of asupplemental nitrogen source, for example ammonium or nitrate.

In another related embodiment, methods of producing an animal feedstockare provided. In certain embodiments, the method comprises culturing arecombinant yeast of the invention in the absence of a supplementalnitrogen source, for example ammonium or nitrate. Advantageously, thesemethods provide an inexpensive source of animal feedstock, as therecombinant yeast provided herein are capable of performing nitrogenfixation and thus can be grown in culture medium that is notsupplemented with a nitrogen source

The yeast of the invention may be genetically modified to furtherenhance the metabolism of particular pentose and or hexose sugars.Exogenous genes encoding for any one of a number of enzymes may beintroduced and expressed in an endophytic yeast used in any one of themethods of the invention. Non-limiting examples of exogenous enzymesthat may be expressed in the yeast of the invention include, xyloseisomerases, xylose reductases, xylose dehydrogenases, NAD-dependentglutamate dehydrogenases, malic enzymes, xylulokinases,phosphoacetyltransferase, aldehyde dehydrogenase, phosphoketolase, andthe like. Examples of the metabolic engineering of yeasts can be found,for example, in Nevoigt, Microbiology and Molecular Biology Reviews 200872(3):379-412.

In certain embodiments, the methods of the invention comprise optimizingthe culture medium in order to maximize the production of a particularproduct, such as ethanol or xylitol.

In another embodiment, the recombinant yeast strains and culturesprovided herein may be useful for fixing atmospheric nitrogen. In aparticular embodiment, the novel strains are useful for fertilizing acrop in the presence or absence of a traditional chemical fertilizer. Inone embodiment, the novel strains are useful for inoculating a crop orcolonizing the soil a crop is planted in with the yeast. The soil may becolonized with the yeast prior to planting the crop, for example before,during, or after tilling the soil in preparation for planning the crop.In other embodiments, the soil may be colonized with the yeast after thecrop has been planted.

V. Xylose Reductase and Xylose Dehydrogenase Polynucleotides andPolypeptides

In one aspect, the present invention provides xylose reductase (XR)polypeptides and xylose dehydrogenase (XDH) polypeptides. In certainembodiments, XR and XDH polypeptides are from a yeast strain selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In some embodiments, thestrain is identified by an rRNA gene sequence selected from any one ofSEQ ID NOS:7 to 18. In one embodiment, the polypeptide has an amino acidsequence that is at least about 85% identical to a an amino acidsequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQID NO:48. In other embodiments, the amino acid sequence of the proteinhas at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to anamino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, and SEQ ID NO:48. In one embodiment, the invention provides apolypeptide encoded by the nucleotide sequence found in FIG. 44, FIG.45, FIG. 46, or FIG. 47. In other embodiments, the amino acid sequenceof the protein has at least about 85%, or at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequenceidentity to an amino acid sequence encoded by the nucleotide sequencefound in FIG. 44, FIG. 45, FIG. 46, or FIG. 47.

In a related aspect, the present invention provides isolated and/orrecombinant polynucleotides encoding for a xylose reductase (XR)polypeptide and/or a xylose dehydrogenase (XDH) polypeptide. In certainembodiments, XR and XDH polypeptides are from a yeast strain selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In some embodiments, thestrain is identified by an rRNA gene sequence selected from any one ofSEQ ID NOS:7 to 18. In one embodiment, the polypeptide encoded by apolynucleotide of the invention has an amino acid sequence that is atleast about 85% identical to a an amino acid sequence selected form SEQID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In otherembodiments, the amino acid sequence of a polypeptide encoded by apolynucleotide of the invention has at least about 85%, or at leastabout 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher sequence identity to an amino acid sequence selected formSEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In oneembodiment, the invention provides a polynucleotide that encodes for apolypeptide encoded by the nucleotide sequence found in FIG. 44, FIG.45, FIG. 46, or FIG. 47. In other embodiments, the invention provides apolynucleotide that encodes for a polypeptide with an amino acidsequence that has at least about 85%, or at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequenceidentity to an amino acid sequence encoded by the nucleotide sequencefound in FIG. 44, FIG. 45, FIG. 46, or FIG. 47.

In a related embodiment, the present invention provides isolated and/orrecombinant polynucleotides comprising an XYL1 and/or XYL2 gene orcoding sequence from an endophytic yeast provided herein. In certainembodiments, the polynucleotide comprises a nucleotide sequence that hasat least 85% sequence identity to a nucleotide sequence selected fromthe group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, andSEQ ID NO:47. In other embodiments, the polynucleotide comprises anucleotide sequence that has at least about 85% identity, or at leastabout 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher sequence identity to an XYL1 or XYL2 gene sequence orcoding sequence provided herein, for example, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, and SEQ ID NO:47. In certain embodiments, thepolynucleotide may further comprise an intron or intronic sequence. Inone embodiment, the polynucleotide comprising an XYL1 and/or XYL2 geneor coding sequence comprises a nucleotide sequence found in FIG. 44,FIG. 45, FIG. 46, or FIG. 47. In certain embodiments, thepolynucleotides of the present invention may further comprise one ormore introns.

In one embodiment, the polynucleotide sequence encodes for a xylosereductase (XR) protein or a xylose dehydrogenase (XDH) protein. Incertain embodiments, the xylose reductase protein is from the WP1 or thePTD3 stain. In a particular embodiment, the heterologous gene sequenceencodes for a polypeptide having at least about 85%, or at least about86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orhigher sequence identity to an amino acid sequence selected form SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.

In certain embodiments, the polynucleotide may comprise a microbialgenome, for example a bacterial or yeast chromosome, or may comprise anexpression vector, a recombinant or artificial microbial chromosome, forexample a bacterial (BAC) or yeast (YAC) chromosome, a bacterialplasmid, a yeast plasmid, a recombinant bacteria phage, a recombinantviral vector, a mammalian expression vector, a baculovirus vector. Inyet other embodiments, the heterologous gene sequence may encode for afusion protein. In another embodiment, the heterologous gene sequencemay encode for a tagged protein, such as a tagged XR or XDH protein. Inyet other embodiments, the polynucleotide may comprise a dual or highorder expression vector that encodes for an XR and an XDH polypeptideprovided herein.

VI. Methods for Biological Nitrogen Fixation and Fertilization of aPlant

In one aspect, the present invention provides methods for the biologicalfixation of nitrogen. In certain embodiments, the methods comprise theuse of an endophytic yeast capable of fixing atmospheric nitrogen.Endophytic yeast useful for nitrogen fixation include, for example,yeast isolated from within the stems of poplar (Populus) trees. Incertain embodiments, these yeast strains are most closely related toRhodotorula graminis or Rhodotorula mucilaginosa species. In aparticular embodiment, the novel strains of the invention are selectedfrom the group consisting of Rhodotorula graminis strain WP1,Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strainPTD3, and Rhodotorula mucilaginosa strain Ad1. In one embodiment of theinvention, the novel endophytic yeast strains contain an rRNA genesequence that is selected from any one of SEQ ID NOS:7 to 18. In aparticular embodiment, an endophytic yeast strain of the invention mayhave an 18 S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In other embodiments, strain of yeast is a recombinant yeast harboring aheterologous gene sequence from an endophytic yeast strain selected fromthe group consisting of Rhodotorula graminis strain WP1, Rhodotorulamucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, andRhodotorula mucilaginosa strain Ad1, wherein said strain is identifiedby an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18.

In certain embodiments, the heterologous gene sequence may be clonedinto a microbial genome, for example a bacterial or yeast chromosome, ormay comprise an expression vector, a recombinant or artificial microbialchromosome, for example a bacterial (BAC) or yeast (YAC) chromosome, abacterial plasmid, a yeast plasmid, a recombinant bacteria phage, arecombinant viral vector, a mammalian expression vector, a baculovirusvector. In yet other embodiments, the heterologous gene sequence mayencode for a fusion protein. In another embodiment, the heterologousgene sequence may encode for a tagged protein, such as a tagged XR orXDH protein.

In certain embodiments, the recombinant yeast strains may be aSaccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, aBrettanomyces, a Torulaspora, an Ascobotryozyma, a Citeromyces, aDebaryomyces, an Eremothecium, a Issatchenkia, a Kazachstania, aKluyveromyces, a Kodamaea, a Kregervanrija, a Kuraishia, a Lachancea, aLodderomyces, a Nakaseomyces, a Pachysolen, a Pichia, a Saturnispora, aTetrapisispora, a Torulaspora, a Vanderwaltozyma, a Williopsis, and thelike. In a particular embodiment, the recombinant yeast is aSaccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, ora Brettanomyces. In one embodiment, the strain is Saccharomycescerevisiae. In a related aspect, biologically pure cultures of therecombinant yeast strains are provided.

In one embodiment, the invention provides a method for fertilizing acrop, the method comprising inoculating the crop with a strain of yeastcapable of fixing nitrogen. In certain embodiments, the step ofinoculating a crop comprises colonizing the soil the crop is planted inwith the yeast. The soil may be colonized with the yeast prior toplanting the crop, for example before, during, or after tilling the soilin preparation for planning the crop. In other embodiments, the soil maybe colonized with the yeast after the crop has been planted.

The methods for fertilizing a crop with a nitrogen fixing yeast providedherein may, in certain instances, be used in conjunction or tosupplement chemical fertilization or alternatively may replace chemicalfertilization. For example, in certain embodiments the present inventionprovides a method for fertilizing a crop comprising inoculating the cropwith a nitrogen fixing yeast in the absence of traditional fertilizer.In other embodiments, a method for fertilizing a crop is provided thatcomprises both inoculating the crop with a nitrogen fixing yeast and theuse of a traditional chemical fertilizer. In certain embodiments, theamount of chemical fertilizer used may be less than would otherwise beused in the absence of a nitrogen fixing yeast, for example, at leastabout 5% less chemical fertilizer, or at least about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 99% less chemical fertilizer than would otherwise be used in theabsence of a nitrogen fixing yeast.

In some embodiments, the crop may be a food crop, including withoutlimitation, sugar cane, maize, wheat, rice, potatoes, sugar beets,soybean, oil palm fruit, barley, tomato, coffee, cocoa, and the like. Incertain embodiments, the crop may be a cereal grain, such as maize,rice, wheat barley, sorgum, millet, oats, rye, triticale, buckwheat,fonio, Quinoa, and the like; a vegetable, a melon, a root, a tuber, afruit, a pulse, and the like.

In other embodiments, the crop may be a non-food crop, including withoutlimitation, a crop grown for the production of a biofuel, such as agrass, a woody plant, a tree or shrub, such as a poplar, willow, orcottonwood, and the like; a crop used for building and or construction,such as hemp, wheat, linseed, flax, bamboo, and the like; a crop usedfor the production of a fiber, such as coir cotton, flax, hemp, manilahemp, papyrus, sisal, and the like; a crop used for the production of apharmaceutical or recombinant protein, such as borage, Echinacea,Artemisia, tobacco, and the like; a crop used for the production of abiopolymer, such as wheat, maize, potatoes, and the like; a crop usedfor the production of a specialty chemical, such as lavender, oilseedrape, linseed, hemp, and the like.

Definitions

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentinvention. For purposes of the present invention, the following termsare defined.

As used herein, the term “endophytic yeasts” refers to fungi thatreproduce asexually by budding from single cells, with absent or reducedhyphal states.

As used herein, “fermentation” refers to a process of breaking downand/or reassembling an organic substance. Fermentation may be eitheraerobic, anaerobic, or partially anaerobic (i.e. in the presence of lowoxygen content). In the case of the present invention, fermentationgenerally refers to the production or conversion of an alcohol or asugar alcohol, such as ethanol or xylitol, from a sugar or mixture ofsugars, including pentose and hexose sugars.

As used herein, a “biologically pure culture” refers to a cultureinoculated with a single microorganism or a sing strain ofmicroorganism. Generally, the microorganism inoculated in a biologicallypure culture may comprise at least about 50% of the total living mass ofsaid culture. In certain embodiments, the microorganism may comprise atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or all of the living mass of a biologically pure culture.

As used herein, a “lignocellulosic biomass” refers to biomass comprisingcellulose, hemicellulose, and lignin. Many sources of lignocellulosicbiomass are used for industrial fermentation, for example, wood residues(e.g., sawmill and paper mill discards), municipal wastes (e.g.,newspaper and paper wastes), agricultural residues (e.g., corn stover,sugarcane bagasse, animal manures, cereal or flax straw, fruit,vegetable, and nut crop), dedicated energy crops (e.g., woody grasses,wood such as willow or poplar, corn, millets, clover), and the like.

The term “nucleic acid molecule” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single-strandedor double-stranded form. It will be understood that when a nucleic acidmolecule is represented by a DNA sequence, this also includes RNAmolecules having the corresponding RNA sequence in which “U” (uridine)replaces “T” (thymidine).

The term “recombinant nucleic acid molecule” refers to a non-naturallyoccurring nucleic acid molecule containing two or more linkedpolynucleotide sequences. A recombinant nucleic acid molecule can beproduced by recombination methods, particularly genetic engineeringtechniques, or can be produced by a chemical synthesis method. Arecombinant nucleic acid molecule may include a protein of interest,such as a protein identified as useful in the production of xylitol orethanol. The term “recombinant host cell” refers to a cell that containsa recombinant nucleic acid molecule. As such, a recombinant host cellcan express a polypeptide from a “gene” that is not found within thenative (non-recombinant) form of the cell.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

Reference to a polynucleotide “encoding” a polypeptide, protein, orenzyme means that, upon transcription of the polynucleotide andtranslation of the mRNA produced therefrom, a polypeptide is produced.The encoding polynucleotide is considered to include both the codingstrand, whose nucleotide sequence is identical to an mRNA, as well asits complementary strand. It will be recognized that such an encodingpolynucleotide is considered to include degenerate nucleotide sequences,which encode the same amino acid residues. Nucleotide sequences encodinga polypeptide can include polynucleotides containing introns as well asthe encoding exons.

The term “expression control sequence” refers to a nucleotide sequencethat regulates the transcription or translation of a polynucleotide orthe localization of a polypeptide to which to which it is operativelylinked. Expression control sequences are “operatively linked” when theexpression control sequence controls or regulates the transcription and,as appropriate, translation of the nucleotide sequence (i.e., atranscription or translation regulatory element, respectively), orlocalization of an encoded polypeptide to a specific compartment of acell. Thus, an expression control sequence can be a promoter, enhancer,transcription terminator, a start codon (ATG), a splicing signal forintron excision and maintenance of the correct reading frame, a STOPcodon, a ribosome binding site, or a sequence that targets a polypeptideto a particular location, for example, a cell compartmentalizationsignal, which can target a polypeptide to the cytosol, nucleus, plasmamembrane, endoplasmic reticulum, mitochondrial membrane or matrix,chloroplast membrane or lumen, medial trans-Golgi cisternae, or alysosome or endosome. Cell compartmentalization domains are well knownin the art and include, for example, a peptide containing amino acidresidues 1 to 81 of human type II membrane-anchored proteingalactosyltransferase, or amino acid residues 1 to 12 of the presequenceof subunit IV of cytochrome c oxidase (see also, Hancock et al., EMBOJ., 10:4033-4039 (1991); Buss et al., Mol. Cell. Biol., 8:3960-3963(1988); U.S. Pat. No. 5,776,689, each of which is incorporated herein byreference).

The term “polypeptide” or “protein” refers to a polymer of two or moreamino acid residues. The terms apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical analogue of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers. The term “recombinant protein” refers toa protein that is produced by expression of a nucleotide sequenceencoding the amino acid sequence of the protein from a recombinant DNAmolecule.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (Ala, A), Serine (Ser, S),Threonine (Thr, T); 2) Aspartic acid (Asp, D), Glutamic acid (Glu, E);3) Asparagine (Asn, N), Glutamine (Gln, Q); 4) Arginine (Arg, R), Lysine(Lys, K); 5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M),Valine (Val, V); and 6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y),Tryptophan (Trp, V).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site at www.ncbi.nlm.nih.gov/BLAST/, or the like). Suchsequences are then said to be “substantially identical” or“substantially similar.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is about50, 100, 200, 300, 400, 500, or more amino acids or nucleotides inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. In certain embodiments, acomparison window may be at least about 25, 50, 75, 100, 150, 200, 250,300, 400, 500, 600, or more positions. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J Mol. Biol., 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA, 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds., Wiley Interscience(1987-2005)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402(1977) and Altschul et al., J. Mol. Biol.215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (see the internet at www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=-4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

A subject nucleotide sequence is considered “substantiallycomplementary” to a reference nucleotide sequence if the complement ofthe subject nucleotide sequence is substantially identical to thereference nucleotide sequence. The term “stringent conditions” refers toa temperature and ionic conditions used in a nucleic acid hybridizationreaction. Stringent conditions are sequence dependent and are differentunder different environmental parameters. Generally, stringentconditions are selected to be about 5° C. to 20° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature, under defined ionic strengthand pH, at which 50% of the target sequence hybridizes to a perfectlymatched probe.

The term “isolated” or “purified” refers to a strain, such as a yeaststrain, or material, such as a protein or nucleic acid, that issubstantially or essentially free from components that normallyaccompany the material in its native state in nature. Purity orhomogeneity generally are determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis, high performanceliquid chromatography, rRNA gene sequencing, and the like. A yeaststrain, polynucleotide, or polypeptide is considered to be isolated whenit is the predominant species present in a preparation. Generally, anisolated protein or nucleic acid molecule represents greater than 80% ofthe macromolecular species present in a preparation, often representsgreater than 90% of all macromolecular species present, usuallyrepresents greater than 95%, of the macromolecular species, and, inparticular, is a polypeptide or polynucleotide that purified toessential homogeneity such that it is the only species detected whenexamined using conventional methods for determining purity of such amolecule. Generally, an isolated yeast strain represents greater than50% of all microbiological species present in a sample, oftentimes anisolated yeast strain will represent greater than 75%, or greater thanabout 80%, 85%, 90%, 95%, of more of all microbiological species presentin a sample.

EXAMPLES Example 1

Isolation of Endophytic Yeast from Poplar Stems

One yeast strain isolated from stems of wild cottonwood (Populustrichocarpa) in Three Forks Park at the Snoqualmie River near the townsof North Bend and Snoqualmie, King County, Wash. was named wild poplarstrain 1 (WP1). Two yeast strains isolated from stems of hybrid poplar(Populus trichocarpa×P. deltoides) in greenhouses at the University ofWashington, Seattle, and Oregon State University, Corvallis were namedas PTD2 and PTD3, respectively. The poplar stems were surface-sterilizedwith 10% bleach (1.2% active sodium hypochloride) for 10 minutes and 1%iodophor for 5 minutes, and then rinsed for 3-5 times with sterilewater. The ends of the explants were removed, and stems were incubatedin the light on Murashige and Skoog medium (MS; Caisson 61 laboratoriesInc., Rexburg, Id.). Morphologically-distinct colonies were streakpurified on YPD (Yeast extract, Peptone, and Dextrose) plates.

Rhodotorula glutinis strain ATCC 2527, obtained from American TypeCulture Collection (ATCC), and a Baker's yeast Saccharomyces cerevisiaeMeyen ex E.C. Hansen strain (Lesaffre yeast corporation, Milwaukee,Wis.) were used for comparison. The phylogenetic relatedness in thethree rRNA genes: 18 S, 26S (D1/D2 domains), and ITS, as well as theirphenotypic characteristics was examined. The capacity to produce IAA bythe three yeast strains was also examined.

FIG. 1 shows photomicrographs of the three yeast strains and R. glutinisATCC in YPD broth. WP1 cells (1A) were ovoid to subspherical,subhyaline, vacuolate, budding on one end, and 6.5-9×5-8 μm. PTD2 cells(1B) were broadly ovoid, subhyaline, vacuolate, budding on one end, and5-7×3-4 μm. Strain PTD3 cells (1C) were ovoid to subspherical,subhyaline, vacuolate, exhibited budding on one end, and 4-5.5×3-4 μm.R. glutinis (ATCC strain) cells (1D) were ellipsoid to ovoid,sub-olivaceous to sub-hyaline, budding at one or both ends, formingpseudohyphae, and 4.5-7.5×3-5 μm. All strains formed colonies of varyingshades of pink on YPD agar plates at 30° C. Sexual reproduction was notobserved in any of the three Populus isolates during culturing forone-week period at 30° C.

Example 2

Extraction of Yeast Genomic DNA

Genomic DNA of yeast was prepared according to the rapid isolation ofyeast chromosomal DNA protocol (Ausubel et al., 1995) withmodifications. Yeast cultures grown overnight in 10 mL YPD broth at 30°C. were collected by centrifuging at 3000×g for 5 min under roomtemperature and washed with 1 mL sterile DI H₂O. Cells were lysed byvortexing with 0.5 g glass beads in 1 mL breaking buffer and 1 mLphenol/chloroform/isoamyl alcohol at high speed for 3 min. The waterlayer was separated by centrifugation, transferred, and washed throughmultiple phenol/chloroform extraction steps. Extracted DNA was thenprecipitated using an equal amount of isopropanol at room temperature.Resuspended DNA in TE buffer was stored at −20° C.

Example 3

PCR Amplification of 18 S, ITS, and D1/D2 Region

The present example focuses on the phylogenetic relatedness of threerRNA genes: 18 S, 26 S (D1/D2 domains), and ITS, from the novel isolatedyeast strains

Yeast DNA was purified and amplified with PCR using three sets ofprimers for 18 S, ITS, and D1/D2 region of rRNA genes, respectively. Theprimers used in this study are listed in Table 1. A 1.8-kb fragment of18 S rRNA gene was amplified with primers NS8 and NS1. A 600-650 bpfragment of D1/D2 region at the 5′ end of the large-subunit rRNA 84 genewas amplified with primers F63 and LR3. A 600-620 bp fragment ofITS1-5.8S-ITS2 region on the rRNA gene was amplified with primers ITS1and ITS4. PCR was performed on DNA extracts in 25 μl with finalconcentrations of 1×PCR Pre-Mix buffer E (Epicentre, Madison, Wis.), 100nM of forward and reverse primers, 5 U of Taq DNA polymerase(Fermentas), and 1 μL of template DNA. The reaction mixture was held at95° C. for 5 minutes followed by 34 cycles of amplification at 95° C.for 30 s, annealing temperature as shown in Table 1 for 30 s and 72° C.for 60 s, with a final step of 72° C. for 5 minutes in a Mastercyclerthermalcycler (Eppendorf, Westbury, N.Y.).

TABLE 1 Primers used for the PCR amplification of 18S, ITS, and D1/D2 genomic rRNA regions. Annealing SEQ temperature, PrimerSequence (5′-3′) ID NO: ° C. Reference NS8 FP TCC GCA GGT TCA CCT ACG GA1 44 White et al., 1990 NS1 RP GTA GTC ATA TGC TTG TCT C 2 44White et al., 1990 F63 FP GCA TAT CAA TAA GCG GAG GAA AAG 3 45Fell et al., 2000 LR3 RP GGT CCG TGT TTC AAG ACG G 4 45Fell et al., 2000 ITS1 FP TCC GTA GGT GAA CCT GCG G 5 44White et al., 1990 ITS4 RP TCC TCC GCT TAT TGA TATG C 6 44White et al., 1990

Example 4

Molecular Cloning and Sequencing

PCR products were subjected to electrophoresis in 0.8% agarose gel.Target bands were collected from the agarose gel and DNA extracted fromit using the QIAEXII gel extraction kit (Qiagen, Madison, Wis.). DNAfragments were cloned using the pGEM T Easy kit (Promega, Madison, Wis.)following the manufacturer's instructions. Sequencing was conductedusing the BigDye Terminator v3.1 Cycle Sequencing kit (AppliedBiosystems) and an ABI 3730XL sequencer (Applied 100 Biosystems) at theDepartment of Biochemistry sequencing facility of the University ofWashington. Sequence data have been submitted to Genbank under theaccession numbers EU563924-EU563932.

TABLE 2 Genomic rRNA sequences. Accession SEQ Strain Gene number ID NO:WP1 18S ribosomal RNA EU563924 7 PTD2 18S ribosomal RNA EU563925 8 PTD318S ribosomal RNA EU563926 9 WP1 ITS ribosomal RNA EU563927 10 PTD2 ITSribosomal RNA EU563928 11 PTD3 ITS ribosomal RNA EU563929 12 WP1 26SD1/D2 ribosomal RNA EU563930 13 PTD2 26S D1/D2 ribosomal RNA EU563931 14PTD3 26S D1/D2 ribosomal RNA EU563932 15

Example 5

Analysis of DNA Sequences

DNA sequences were aligned with the program ClusterW (Thompson et al.,1994) using default gap penalties. The selection of sequences forconstruction of phylogenetic trees was done by comparing the targetsequences to all sequences in the GenBank by the online BLAST program.Phylogenetic trees were constructed using the neighbor-joining distancemethod (Saitou and Nei, 1987) and distances computed using theJukes-Cantor methods (Jukes and Cantor, 1969) and using the MaximumParsimony method (Eck and 111 Dayhoff, 1966). All analyses wereconducted with the program MEGA 4 (Tamura et al., 2007).

Analysis of the 18 S, ITS1-5.8S-ITS2, and D1/D2 regions suggested thatisolates PTD2 and PTD3 were most closely related to Rhodotorulamucilaginosa (FIGS. 2-7). PTD2 was identical to R. mucilaginosa in boththe 18 S and D1/D2 region sequences, but differed from R. mucilaginosain 5 of 183 base positions when the ITS1-5.8S-ITS2 sequences werecompared. PTD3 was identical to R. mucilaginosa in the D1/D2 region, anddiffered from R. mucilaginosa in 1 of 586 bases in the 18 S sequence and7 of 583 bases in the ITS1-5.8S-ITS2 sequence.

Sequences from WP1 appeared to support relationships with severaldifferent species. Rhodosporidium babjevae and WP1 shared the mostsimilar 18 S rRNA gene sequences (FIG. 2), while the ITS1-5.8S-ITS2 andD1/D2 sequences of WP1 were most similar to those of Rhodotorulagraminis and Rhodotorula glutinis (FIGS. 3 and 4), respectively. TheITS1-5.8S-ITS2 sequence of WP1 differed from R. glutinis at 4 of 583base positions (all in the ITS1 region), from R. graminis in 1 of 583base positions (in the ITS1 region), and from that of R. babjevae in 6of 583 positions (4 in ITS1, 1 in 5.8S, and 1 in ITS2). In the D1/D2region, WP1 was identical to R. glutinis based on 586 positions, anddiffered from R. graminis in 1 and from R. babjevae in 2 base positions.In the 18 S, WP1 was identical to R. babjevae based on 952 positions,and differed from R. glutinis by 1/952. It should be noted that the WP118 S sequence was identical to the sequence of a R. graminis strain inGenBank (Accession number X83827) but the R. graminis sequence containedmissing data for 7 positions. The strain was not included in thephylogenetic tree shown in FIGS. 2 and 5.

Example 6

IAA Production Test

To quantify the production of IAA, isolates were grown in YPD/YMA mediumwith or without 0.1% (w/v) L-tryptophan for 1, 2, 5, and 7 days and 1.5mL of the cells were pelleted by centrifugation at 10,000×g for 5 min.One mL of supernatant was mixed with 2 mL of Salkowski reagent (2 mL of0.5 M FeCl₃+98 mL 35% HClO₄) (Gordon and Weber, 1951), and the intensityof pink color developing in the mixture after 30 min was quantified by aHach DR/4000 spectrophotometer (Hach, Loveland, Colo.) at wavelength 530nm. Cell pellets were dried at 100° C. overnight and weighed fornormalizing IAA production. Similarly, pink color was also developed fora series of IAA standard solutions to establish a stand curve.

No detectable IAA was produced for all tested yeast strains after 7-dayincubation without L-tryptophan. When incubated with 0.1% L-tryptophan,strains of WP1, PTD2, PTD3, and R. glutinis ATCC showed significantproduction of IAA (FIG. 9). No detectable IAA was produced by Baker'syeast. The overall production of IAA increased with time for the fouryeast strains. Among them, WP1 had the highest IAA production and PTD3had the least

Example 7

Phenotypic Characterization of Yeast Strains

The morphology of yeast strains was determined and photographs madeusing a Leica DMR compound microscope equipped with brightfield anddifferential interference contrast optics and a Leica DC300 digitalcamera (Leica Microsystems GmbH, Wetzlar). Utilization of kinds ofcarbon sources was examined using a commercial API 20C AUX yeastidentification kit (bioMerieux, Durham, N.C.) according to themanufacture's instructions. Yeast cultures in YPD broth diluted to anoptical density (OD₆₀₀) of 0.451, which is equivalent to McFarlandstandard No. 2, in 0.85% NaCl solution were applied to the API 20C AUXstrips. The strips then were incubated at 30° C. Pink color developed onthe incubation strips at 48 and 72 hours to indicate utilization ofindividual carbon sources by tested yeast strains.

A commonly used method to distinguish many yeast species is comparisonof their abilities to utilize certain organic compounds as the solemajor source of carbon (Barnett et al., 2000). A commercial yeastidentification kit, API 20C AUX, can identify rapidly common and rareclinical yeast isolates with high efficacy (Ramani et al., 1998; Verweijet al., 1999). Table 3 summarizes utilization of 19 different organiccompounds by the three Populus isolates and two controls (R. glutinisATCC and Baker's yeast), with the API 20C AUX system. Characteristics ofR. graminis were compiled from the literature (Barnett et al. 2000).

TABLE 3 Summary of utilization of 19 carbon sources by strains WP1,PTD2, PTD3, ATCC, and Baker's yeast assessed using the API 20C AUXsystem. R. glutinis Baker's Carbon Source WP1 PTD2 PTD3 ATCC yeast R.graminis D-glucose + + + + + + Glycerol + + − − − + Calcium + − − + − V2-keto- gluconate L-arabinose − + + − − +, D D-xylose + + + − − +Adonitol + + + − − NA Xylitol − + + − − +, D D-galactose + − + + + +Inositol − − − − − − D-sorbitol + + + + − NA Methyl- − − − − + NAαD-gluco- pyranoside N-acetyl- − − − − − − glucosamine D-cellobiose − −− − − + D-lactose − − − − − − D-maltose − + + + + V Sucrose + + + + + +D-trehalose − + + − + +, D D-melezitose − V − + + − D-raffinose + + + +− + The profile for R. graminis was compiled from Barnett et al. (2000).“+”—positive, “−”—negative, “V”—variable, “NA”—not available,“D”—delayed longer than 7 days

The clusterings between Baker's yeast and S. cerevisiae and between R.glutinis ATCC and R. glutinis in FIG. 8 demonstrated that theidentification system was effective. Similarity of PTD2, PTD3, and R.mucilaginosa (FIG. 8) carbon utilization profiles confirmed thegroupings based on rRNA gene sequences. However, WP1 did not clusterwith any Rhodotorula species in the reference list of the API 20C AUXsystem (FIG. 8) and diverged in utilization of 5 compounds (glycerol,D-xylose, adonitol, D-maltose, and D-melezitose) when compared with R.glutinis ATCC. Compared to the reported carbon-utilization profile fromthe literature, WP1 diverged from R. graminis only in utilizingD-cellobiose out of 13 organic compounds. Regarding another 3 compounds(L-arabinose, xylitol, and D-trehalose) labeled positive for R. graminisin Table 2 with over 7-day delayed observation, direct comparison to theWP1 profile obtained after 3-day incubation with API 20C AUX system isnot proper.

Example 8

Clustering Analysis of Phenotypic Characteristics of Yeast Strains

A clustering map was drawn by JMP statistics software version 6 (SAS,Cary, N.C.) according to the Ward's minimum variance method (Milligan,1980). Distance for Ward's method is determined according to theformula:

$D_{KL} = \frac{{{{\overset{\_}{x}}_{K} - {\overset{\_}{x}}_{L}}}^{2}}{\frac{1}{N_{K}} + \frac{1}{N_{L}}}$wherein X_(K) and X_(L) are the mean vectors for cluster C_(K) andC_(L), respectively, C_(K) is the K^(th) cluster and C_(L) is the L^(th)cluster; N_(K) and N_(L) are the numbers of observations in C_(K) andC_(L).

Three pink-pigmented yeast strains isolated from stems of Populus grewwell on YPD medium under aerobic conditions. Phylogenetic analysis ofrRNA gene sequences supported determination of the yeast strains, PTD2and PTD3 as Rhodotorula mucilaginosa. Determination of WP1 was not assimple as that of PTD2 and PTD3 since analyzing different sequence dataprovided differing results. Rhodotorula and Rhodosporidium are membersof the class Urediniomycetes of phylum Basidiomycota. Rhodotorulaglutinis, R. graminis, R. babjevae, and R. mucilaginosa grouped togetherin the Sporidiobolus clade, based on phylogenetic analysis of ITS andD1/D1 regions (Fell et al., 2000; Scorzetti et al., 2002). Rhodotorulaglutinis, R. graminis, and R. babjevae occurred on the same branch ofthe Sporidiobolus clade, suggesting a close phylogenetic relationshipamong them. As the ITS region is generally considered to be lessconserved than either small or large subunits of rRNA genes (Scorzettiet al., 2002), the ITS analysis could be more informative indistinguishing close related species. A single substitution out of 583positions in the ITS1-5.8S-ITS2 between WP1 and R. graminis compared to4 substitutions for WP1 and R. glutinis and to 6 substitutions for WP1and R. babjevae suggests that WP1 is more closely related to R.graminis. In addition, WP1 shared higher similarity oncarbon-utilization profiles with R. graminis than R. glutinis. Based onthe phylogenetic and phenotypic characteristics of WP1, we regard theWP1 isolate as most closely fitting the current concept of R. graminis.

Rhodotorula mucilaginosa has been isolated from a wide variety ofsources, including the bark of Quercus suber L. (cork oak)(VIIIa-Carvajal et al., 2004), soil and mosses from Antarctica (Pavlovaet al., 2001), food stuffs (Haridy, 1993; Botes et al., 2007), andhumans (Neofytos et al., 2007). The species has been reported frequentlyfrom wastewater treatment plants and exhibited tolerance to heavy metalssuch as copper, cadmium, and uranium (de Siloniz et al., 2002;Balsalobre et al., 2003; Villegas et al., 2005). Epoxide hydrolase of R.mucilaginosa can hydrolyze glycidyl ethers (Kotik et al., 240 2005),dibenzofuran (Romero et al., 2002), and other benzene compounds(Middelhoven 241 et al., 1992) in environmental bioremediationprocesses. Rhodotorula graminis was first isolated from the leafsurfaces of pasture grasses (di Menna, 1958) and later found widely inthe environment, being isolated from soil (Vadkertiova and Slavikova,1994; Hobbie et 244 al., 2003), Ceratonia siliqua L. (carob trees)(Spencer et al., 1995), and tropical fruits (Trindade et al., 2002). Thespecies has shown an ability to cleave aromatic rings (Durham et al.,1984) and has been studied for the bioremediation of benzene compounds(Middelhoven, 1993).

Recently, the role of endophytes in phytoremediation of xenobiotics hasbeen highlighted, including increasing plant tolerance to heavy metals(Lodewyckx et al., 2001), reducing phytotoxicity of herbicides (Germaineet al., 2006), and facilitating degradation of nitro-aromatic compounds(van Aken et al., 2004b). The tolerance to heavy metals and degradationof xenobiotics by R. mucilaginosa and R. graminis suggests the newPopulus endophytic yeast strains may be suitable for phytoremediationapplications. Furthermore, the production of IAA by the three yeaststrains could potentially promote plant growth.

The three yeast strains produced IAA only with the addition ofL-tryptophan. As one of the most expensive standard protein amino acids,in terms of energy, to produce (Hrazdina and Jensen, 1992), tryptophanis not biosynthesized by all bacteria and yeasts. Those microorganismsincapable of synthesizing tryptophan have to rely on their plant hostsor surrounding microbial sources (Radwanski and Last, 1995). Withtryptophan available in the Populus tissue, the endophytic yeasts do nothave to spend high energy on synthesis of the amino acid by themselves.At the same time, the ability to convert tryptophan to IAA by theendophytes would, in return, benefit the tryptophan provider, which maybe seen as a mutually advantageous plant-microbe example.

To our knowledge, the yeast strains provided by the present inventionare the first endophytic yeast strains isolated from species of Populus.The strain from wild Populus, WP1, has been chosen for whole genomesequencing by the Joint Genome Institute of the Department of Energy dueto its potential applications for bioenergy production. Thedetermination and characterization presented in the present inventionshould benefit future research on these strains.

Example 8

Growth Requirement Test

In order to study the sugar utilization of the endophytic yeast strains,WP1, PTD3 and the baker's yeast (BK), isolates were streaked from frozenglycerol stocks onto yeast extract, peptone, dextrose (YPD) agar toobtain isolated colonies. A single colony was transferred to 10 ml ofYPD broth and incubated on a shaker at 30° C. overnight. The overnightculture was harvested and washed with MS medium (Caisson Labs MSP009)twice. For growth curve assays, cells were grown in 25 ml of MS mediumcontaining either 3% glucose or 3% xylose at pH 5.8. Growth wasmonitored using a spectrophotometer measuring the optical density at 600nm (OD600). Statistical analysis was done using split plot ANOVA(Intercooled Stata 10.0, StataCorp LP, College Station, Tex.) in orderto account for the multiple measures taken over time on each flask, andthe replicated flasks for each sample.

In order to study the sugar utilization of the two endophytic yeaststrains, WP1 and PTD3, growth rate was monitored in media with differentsugars. The growth curve experiments showed that both WP1 and PTD3 grewwell in glucose (FIG. 34A) and xylose (FIG. 34B) sugars. As reportedpreviously, Baker's yeast did not utilize xylose. It is also noteworthythat PTD3 grew better than WP1 under the two conditions and PTD3 was abetter xylose utilizer (FIG. 34B). There was about a 24 hour-delaybefore WP1 and PTD3 started growing in glucose and xylose. The delay wasmost likely from the shift from rich medium (YPD) to minimal medium(plain MS).

Example 9

Cloning of the Xylose Reductase (XR) and Xylitol Dehydrogenase (XDH)Encoding Genes XYL1 and XYL2 from WP1

Yeast strain WP1 was isolated from stems of wild cottonwood (Populustrichocarpa) and was identified as Rhodotorula graminis (Xin et al.2009). Another yeast strain, PTD3, was isolated from stems of hybridpoplar (Populus trichocarpa×P. deltoides) and was identified to bespecies Rhodotorula mucilaginosa (Xin et al. 2009). A baker's yeastATCC6037 strain was used as the control yeast.

WP1 and PTD3 genomic DNA was prepared following a published protocol(Burke et al. 2000) with the following modifications: two extraphenol:chloroform/chloroform extractions and isopropanol precipitationwere carried out. For mRNA preparation, cells were grown in YPD, whichwas prepared as described (Kaiser et al. 1994) except that sugars wereautoclaved separately from the basal medium. YPX and YPGX were similarto YPD but replaced dextrose with xylose or xylose plus glucose.Isolation of mRNA was performed by the method described in (Laplaza etal. 2006).

Isolated RNA was quantified using a NanoDrop spectrophotometer (ND1000).Reverse transcription (RT) and subsequent PCR amplifications wereperformed sequentially using the OneStep RT-PCR Kit (QIAGEN). The wholeWP1 XR and XDH-encoding genes were amplified by RT-PCR using two sets ofprimers (WP1-XR-F, WP1-XR-R and WP1-XDH-F, WP1-XDH-R), which weredesigned based on the sequences of XYL1 and XYL2 genes in Pichiastipitis (GenBank accession numbers: CAA42072, AAD28251) as well as thealignment results with WP1 whole genome sequence (sequencing by JGI andis available online) with the following modifications.

The genome sequence of WP1 was provided through the DOE Joint GenomeInstitute sequencing effort (see Acknowledgements). Putative XYL1 andXYL2 genes were first found in the JGI sequence using BLAST and theresulting sequences were utilized to design primers for the cloning ofthe mRNA sequences of the two genes from WP1. Sequence comparisons ofthe cloned genes with public databases were performed via the Internetat the National Center for Biotechnology Information site (see theinternet at www.ncbi.nlm.nih.gov/), by employing the tblast algorithm(Altschul et al. 1997). GenomeScan (Chris Burge, Biology Dept. at MITsee the internet at genes.mit.edu/genomescan.html) was employed topredict the gene exon/intron structures and putative XR and XDH mRNAsequences in WP1. All the resulting sequences in WP1 and PTD3 werealigned with homologous protein sequences of other D-xylose-fermentingyeasts (e.g. Pichia stipitis, Candida. spp) using the local BLASTprogram (B12seq).

The resulting PCR products were purified using the QIAEXII gelextraction kit (Qiagen, Madison, Wis.) and then inserted into the pGEM-TEasy vector (Promega, Madison, Wis.) following the manufacturer'sinstructions. Sequencing of the inserts in both directions was performedby the UW Biochemistry Department Sequencing Facility using the BigDyeTerminator v3.1 Cycle sequencing kit (Applied Biosystems).

TABLE 4 Primers used for cloning of the XR andXDH-encoding genes from WP1 and PTD3 and in expression studies thereof.SEQ ID Primer Sequence NO: WP1-XR-F ATGGTCCAGACTGTCCCC 19 WP1-XR-RTCAGTGACGGTCGATAGAGATC 20 WP1-XDH-F ATGAGCGCTCCCAGTCTCGC 21 WP1-XDH-RTCACTCGAGCTTCTCGTCGAC 22 PTD3-D-XR-F GCYATCAAGKCGGGYTACCG 23 PTD3-D-XR-RGTGGWAGBTGTTCCASAGCTT 24 PTD3-D-XDH-F CCMATGGTCYTSGGNCACGA 25PTD3-D-XD-R CCGACVGGVCCDGCDCCAAAGAC 26 PTD3-XR-GSP1 GCCAGTGGATGAGGTAGAGG27 (for 5′ RACE) PTD3-XR-GSP2(for GTGATGAAGATGTCCTTGCG 28 5′ RACE)PTD3-XR-GSP3(for AGGTCTACGGCAACCAGAAG 29 3′ RACE) PTD3-XR-GSP4(forATCACCTCGAAGCTCTGGAAC 30 3′ RACE) PTD3-XDH-GSP1 GATGAGCGATTTGAGGTTGAC 31(for 5′ RACE) PTD3-XDH-GSP2 CCTTGGCAACTGCGTGGAC 32 (for 5′ RACE)PTD3-XDH-GSP3(for GCAAAGGTGGTCATTACGAAC 33 3′ RACE) PTD3-XDH-GSP4(forCTCCTTGAGCCCATGTCGGT 34 3′ RACE) #XR-F ATCACCTCGAAGCTCTGGAAC 35 #XR-RGCCAGTGGATGAGGTAGAGG 36 #XDH-F CTCCTTGAGCCCATGTCGGT 37 #XDH-RGATGAGCGATTTGAGGTTGAC 38 515F(18S rRNA) GTGCCAAGGCAGCCGCGGTAA 391209R(18S rRNA) GGGCATCACAGACCTG 40 note: K = G/T V = A/C/G M = A/C N =A/C/G/T R = A/G B = C/G/T S = C/G W = A/T Y = C/T D = A/T/G

The XR and XDH-encoding genes were cloned and sequenced from WP1 usingprimers based on the genomic sequence of WP1 provided by the DOE JGIsequencing project. Analysis of the two genes was then performed on thecloned sequences (not directly from the JGI sequences provided). The1259 nucleotide sequence of WP1-XR contains an open reading frame of 966nucleotides (SEQ ID NO:41) encoding a polypeptide of 321 amino acids(SEQ ID NO:42). The 1216 nucleotide sequence of WP1-XDH contains an openreading frame of 1191 nucleotides (SEQ ID NO:43) encoding a polypeptideof 396 amino acids (SEQ ID NO:44). At the amino acid level, the WP1-XRgene is 37% and 36% identical to XYL1gene of Pichia stipitis(XP_001385181) and Candida guilliermondii (094735), respectively; theWP1-XDH gene is slightly more conserved: 41% identity to XYL2 gene ofPichia stipitis (XP_001386982) and Candida tropicalis. The visualizedannotation pictures (by using vector NTI10) of the two genes show thatboth XR and XDH genes are more complex than those of Pichia stipitiswhich has no introns in the genes (Figured 35 and 36)(Amore et al.1991).

Example 10

WP1 XR and XDH Gene Expression Levels in Glucose and Xylose

To investigate the XR and XDH gene expression in WP1, cells were grownin medium containing either glucose or xylose, and the RNA was purifiedfrom the cultures. Segments from the mRNA were amplified using RT-PCRwith primers specific for each of the two genes. As shown in FIG. 37,the XR and XDH genes were expressed in WP1 cells grown in xylose. Theseresults indicated that the genes are indeed transcribed and that XR andXDH gene expression was upregulated by xylose. The XR gene expressionwas not detectable when the cells were grown in glucose; however, therewas some low-level constitutive expression of the XDH in glucose.

Briefly, total RNA was isolated from cells grown in media containing 2%glucose, 2% xylose, 1% glucose+1% xylose or 2% glucose+2% xyloserespectively. RT-PCR was applied on the same amount of total RNAs fromdifferent media using the primer sets #XR-F, #XR-R and #XDH-F, #XDH-R(Table 4) designed to work equally well for both WP1 and PTD3. To verifythat the same amount of total RNA was used, 18 S rRNA semiquantitativeRT-PCR was performed in WP1 under these different culture conditionsusing primer set 515F and 1209R (downloaded from JGI for eukaryotic 18 SrRNA gene amplification).

Example 11

Cloning of the Xylose Reductase (XR) and Xylitol Dehydrogenase (XDH)Encoding Genes XYL1 and XYL2 from PTD3

For cloning the partial XR and XDH-encoding genes in PTD3 (genomesequences are not available), RT-PCR was performed using the degenerateprimers PTD3-D-XR-F, PTD3-D-XR-R and PTD3-D-XDH-F PTD3-D-XDH-R, whichwere designed based on the multiple sequence alignment amongst PTD3, WP1and other D-xylose-fermenting yeasts (CLUSTALW, Thompson et al. 1994).Following RT-PCR, samples were subjected to electrophoresis in a 1%agarose get, using Sybersafe (Invitrogen) as a DNA intercalating andvisualizing agent, at 100V for 1 hour.

Since strain PTD3 was a more effective utilizer of xylose compared toWP1, the xylose metabolism genes where cloned from this strain. However,the PTD3 genome has not been sequenced, so a different approach was usedto clone the two genes than was used for WP1. The partial PTD3 XR andXDH-encoding genes were cloned using degenerate primer sets that weredesigned based on the multiple sequence alignment amongst PTD3, WP1 andother D-xylose-fermenting yeasts (Table 4). The complete nucleotidesequences were subsequently determined by 5′ and 3′ rapid amplificationof cDNA ends (RACE) using gene specific primers based on the cDNAfragment sequences. Briefly, the partial PTD3 XR and XDH-encoding geneswere amplified by RT-PCR and sequenced, and the complete nucleotidesequences were subsequently determined by 5′ and 3′ rapid amplificationof cDNA ends (RACE) using a 5′/3′ RACE kit (FirstChoice RLM-RACE Kit,Applied Biosystems). For 5′RACE, the gene-specific primers PTD3-XR-GSP1,PTD3-XR-GSP2, PTD3-XDH-GSP1 and PTD3-XDH-GSP2 were used. For 3′ RACE,the gene-specific primer PTD3-XR-GSP3, PTD3-XR-GSP4, PTD3-XDH-GSP3 andPTD3-XDH-GSP4 were used. Primer sequences are listed in Table 4.

The 1087 bp nucleotide sequence of the cloned PTD3-XR contained an openreading frame of 975 bp nucleotides (SEQ ID NO:45) encoding apolypeptide of 324 amino acids (SEQ ID NO:46). The alignment resultsshow that PTD3-XR protein is 67% identical to the WP1 XR protein (Table5). The 1409 bp nucleotide sequence of PTD3-XDH contains an open readingframe of 1185 bp nucleotides (SEQ ID NO:47) encoding a polypeptide of394 amino acids (SEQ ID NO:48). The alignment results showed thatPTD3-XDH protein is 69% identical to the WP1 XDH protein. Alignmentswith other yeasts were also performed to study the homology with the twogenes in PTD3 (Table 5). The XR and XDH proteins of WP1 and PTD3 were69-73% identical, whereas they are only 37-41% identical to theseproteins from other known xylose-utilizing species.

TABLE 5 XR and XDH identities between homologous proteins in severalyeast strains. Pichia Candida Candida Identity WP1 stipitisguilliermondii tropilis PTD3 XR 73% 38% 37% 39% PTD3 XDH 69% 37% Null41% GenBank Accession No.: XR: CAA42072 (P. stipitis); ABX60132 (C.tropicalis); AAD09330 (C. guilliermondii). XDH: AAD28251 (P. stipitis);ABB01368 (C. tropicalis). The XDH protein sequence of Candidaguilliermondii was unavailable.

Example 12

PTD3 XR and XDH Gene Expression Levels in Glucose and Xylose

To investigate the expression of the two genes in PTD3, cells were grownin glucose and xylose media as with the WP1 study. PTD3 gene specificprimers were used to amplify the segments from mRNA by using RT-PCR.Different bands corresponding to XR and XDH were observed in mRNA fromcells grown on either glucose or xylose (FIG. 38). These resultsindicate that the genes are indeed transcribed within mRNA and that theXR and XDH gene expression was induced by xylose. As in WP1, the geneswere barely expressed in medium containing only glucose as the carbonsource.

Example 13

Comparison of the Gene Expression Levels of XR and XDH Between WP1 andPTD3

In order to better understand the differences in utilization of xylosebetween the two endophytic yeast strains, the expression levels of theXR and XDH genes were compared between the strains. Using the alignedWP1 and PTD3 sequences, primers were designed to the gene regions ofidentity so that the expression of XR and XDH-encoding genes could bedirectly comparable. RT-PCR was performed to amplify the mRNA segmentsfrom WP1 and PTD3 cells grown in YP medium containing different sugars(2% glucose, 2% xylose, 1% glucose+1% xylose, 2% glucose+2% xylose). Asshown in FIG. 39, both the XDH and XR genes are expressed to higherlevels in PTD3 than in WP1 when the yeast were grown in xylose medium.The expression of the two genes appeared slightly suppressed in 1%xylose+1% glucose medium compared to 2% xylose medium.

In order to investigate whether the expression differences resulted fromthe lower xylose concentration in the mixed sugar medium or fromrepression by glucose, an RT-PCR experiment was also conducted under 2%glucose+2% xylose culture condition. As shown in (FIG. 40) in WP1, theexpression of the two genes were still slightly suppressed in 2%xylose+2% glucose medium compared to 2% xylose medium. However, the geneexpression was not suppressed by glucose in PTD3. In this strain, thelevel of the XR and XDH gene expression was about equal in both themixed sugar medium and the xylose medium. 18 S rRNA RT-PCR was performedas an internal control showing that equal amounts of total RNA were usedunder these different culture conditions (FIG. 41).

Example 14

Nitrogen Fixation of Endophytic Yeast

To determine if any of the isolated endophytic yeast strains could fixatmospheric nitrogen, several isolated strains were incubated innitrogen limiting media (NFM). Surprisingly, it was found that WP1, aswell as two other pink yeasts isolated from greenhouse-grown poplarhybrids, were among the endophytes that grew well on NFM. Amplificationof the nifH gene using universal primers indicated that these isolatescontain the nitrogenase gene required for nitrogen fixation. FIG. 33shows the growth of WP1 and Saccharomyces cerevisiae (baker's yeast) inNFM as quantified by OD600. These results suggest that the isolatedendophytic yeast strains provided herein are able to fix atmosphericnitrogen.

Example 15

Use of Endophytic Yeast for Nitrogen Fixation and Supplementation ofNitrogen Deficiencies for Plant Growth

To determine if nitrogen fixing yeast could be used to promote plantgrowth under nitrogen limiting conditions, corn was grown for 11 weeksin soil without nitrogen supplementation in the presence (WP1) orabsence (non-symbiotic; NS) or the nitrogen fixing strain WP1. As seenin FIG. 42, corn grown in the presence of the WP1 yeast strainconsistently grew much more robustly, providing about 5 times morebiomass (B(g)) than corn grown in the absence of WP1 (compare FIG. 42Bwith FIG. 42A, respectively). In addition, the % viability in WP1colonized plants (58-92%) was higher than uninoculated plants(8.3-29.2%) plants. Statistical analysis indicated significantdifferences (P≦0.1) for both viability and biomass with WP1 symbioticplants having higher viability and biomass compared to uninoculatedplants. A graphic representation of these data are provided in FIG. 44.Thus, nitrogen fixing endophytic yeast strains isolated from withinpoplar trees can significantly promote the growth of corn, even in theabsence of traditional nitrogen sources. As such, these yeast can beused for biological nitrogen fixation instead of chemical fertilizers tolower costs and reduce nitrous oxide emissions into the atmosphere.

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A recombinant yeast strain capable of fermentingboth a five carbon sugar and a six carbon sugar, the yeast harboring aheterologous gene sequence from an endophytic yeast strain selected fromthe group consisting of Rhodotorula graminis strain WP1, Rhodotorulamucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, andRhodotorula mucilaginosa strain Ad1, wherein said endophytic yeaststrain is identified by an rRNA gene sequence of SEQ ID NO:9.
 2. Therecombinant yeast strain of claim 1, wherein the heterologous genesequence encodes for a polypeptide comprising an amino acid sequencethat is at least 85% identical to an amino acid sequence selected fromSEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 3. Therecombinant yeast strain of claim 1, wherein the heterologous genesequence encodes for a polypeptide comprising an amino acid sequencethat is at least 90% identical to an amino acid sequence selected fromSEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 4. Therecombinant yeast strain of claim 1, wherein the heterologous genesequence encodes for a polypeptide comprising an amino acid sequencethat is at least 95% identical to an amino acid sequence selected fromSEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 5. Therecombinant yeast strain of claim 1, wherein the yeast is aSaccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, ora Brettanomyces strain.
 6. The recombinant yeast strain of claim 5,wherein the yeast is Saccharomyces cerevisiae.